Metabolomic evolution of the postpartum dairy cow uterus.

High rates of early pregnancy loss are a critical issue in dairy herds, particularly in seasonal, grazing systems. Components of the uterine luminal fluid (ULF), on which the early embryo depends for sustenance and growth, partly determine early pregnancy losses. Here, changes in ULF from early to mid-postpartum in crossbred dairy cows were explored, linking them with divergent embryo development. For this, the uteri of 87 cows at Day 7 of pregnancy at first and third estrus postpartum were flushed to collect ULF. Eighteen metabolites (chiefly organic acids and sugars) significantly varied in abundance across postpartum, indicating a molecular signature of physiological recovery consistent of the upregulation of pyrimidine metabolism and glycerophospholipid metabolism, and downregulation of pentose phosphate and taurine metabolism pathways. Joint pathway analysis of metabolomics data and a previously generated proteomics data set on the same ULF samples suggests key links between postpartum recovery and subsequent successful embryo development. These include upregulation of VEGFA and downregulation of metabolism, NRF2, T-cell receptor, which appear to improve the ULF's capacity of sustaining normal embryo development, and a putative osmo-protectant role of beta-alanine. These relationships should be further investigated to develop tools to detect and reduce early pregnancy loss in dairy cows.

Proteomic determinants of uterine receptivity for pregnancy in early and mid-postpartum dairy cows†.

Dairy cow subfertility is a worldwide issue arising from multiple factors. It manifests in >30% early pregnancy losses in seasonal pasture-grazed herds, especially when cows are inseminated in the early post-partum period. Most losses occur before implantation, when embryo growth depends on factors present in maternal tract fluids. Here we examined the proteomic composition of early and mid-postpartum uterine luminal fluid (ULF) in crossbred lactating dairy cows to identify molecular determinants of fertility. We also explored changes in ULF from first to third estrus cycles postpartum in individual cows, linking those changes with divergent embryo development. For this, we flushed uteri of 87 cows at Day 7 of pregnancy at first and third estrus postpartum, recovering, and grading their embryos. Out of 1563 proteins detected, 472 had not been previously reported in this fluid, and 408 were predicted to be actively secreted by bioinformatic analysis. The abundance of 18 proteins with roles in immune regulation and metabolic function (e.g. cystatin B, pyruvate kinase M2) was associated with contrasting embryo quality. Matched-paired pathway analysis indicated that, from first to third estrus postpartum, upregulation of metabolic (e.g. creatine and carbohydrate) and immune (e.g. complement regulation, antiviral defense) processes were related to poorer quality embryos in the third estrus cycle postpartum. Conversely, upregulated signal transduction and protein trafficking appeared related to improved embryo quality in third estrus. These results advance the characterization of the molecular environment of bovine ULF and may aid understanding fertility issues in other mammals, including humans.

Proteomics and metabolomics in cow fertility: a systematic review.

Cow subfertility is a multi-factorial problem in many countries which is only starting to be unravelled. Molecular biology can provide a substantial source of insight into its causes and potential solutions, particularly through large scale, untargeted omics approaches. In this systematic review, we set out to compile, assess and integrate the latest proteomic and metabolomic research on cow reproduction, specifically that on the female reproductive tract and early embryo. We herein report a general improvement in technical standards throughout the temporal span examined; however, significant methodological limitations are also identified. We propose easily actionable avenues for ameliorating these shortcomings and enhancing the reach of this field. Text mining and pathway analysis corroborate the relevance of proteins and metabolites related to the triad oxidative stress-inflammation-disease on reproductive function. We envisage a breakthrough in cattle reproductive molecular research within the next few years as in vivo sample techniques are improved, omics analysis equipment becomes more affordable and widespread, and software tools for single- and multi-omics data processing are further developed. Additional investigation of the impact of local oxidative stress and inflammation on fertility, both at the local and systemic levels, is key towards realising the full potential of this field.

On the enigmatic disappearance of Rauber's layer.

The polar trophoblast overlays the epiblast in eutherian mammals and, depending on the species, has one of two different fates. It either remains a single-layered, thinning epithelium called "Rauber's layer," which soon disintegrates, or, alternatively, it keeps proliferating, contributing heavily to the population of differentiating, invasive trophoblast cells and, at least in mice, to the induction of gastrulation. While loss of the persistent polar trophoblast in mice leads to reduced induction of gastrulation, we show here that prevention of the loss of the polar trophoblast in cattle results in ectopic domains of the gastrulation marker, BRACHYURY This phenotype, and increased epiblast proliferation, arose when Rauber's layer was maintained for a day longer by countering apoptosis through BCL2 overexpression. This suggests that the disappearance of Rauber's layer is a necessity, presumably to avoid excessive signaling interactions between this layer and the subjacent epiblast. We note that, in all species in which the polar trophoblast persists, including humans and mice, ectopic polar trophoblast signaling is prevented via epiblast cavitation which leads to the (pro)amniotic cavity, whose function is to distance the central epiblast from such signaling interactions.

In Vitro Culture of Deer Embryos.

In vitro embryo production of deer species has the potential to increase valuable traits for the agricultural sector, and from a conservation perspective, it is a propagation tool which can improve genetic diversity in small captive populations. In vitro embryo production is a multistep process consisting of oocyte maturation, fertilization, and embryo culture. These techniques provide the backbone for more advanced assisted reproductive technologies such as intracytoplasmic sperm injection (ICSI), somatic cell nuclear transfer (SCNT), a source of embryonic stem cells, and embryos for gene editing. In vitro-produced embryos are a readily available resource for comparative embryology studies and a functional assay to assess oocyte competence and evaluate in vitro embryo requirements during culture. A semidefined fertilization and culture medium system, deer synthetic oviduct fluid (DSOF), has been formulated based on deer oviduct fluid. Red deer calves (Cervus elaphus) and Thamin Eld's deer fawn (Rucervus eldii thamin) have been produced after the transfer of in vitro embryos (IVF and SCNT) grown in DSOF culture. Here we describe the in vitro method of maturation, fertilization, and embryo culture for deer species.

MicroRNA expression in bovine preimplantation embryos.

We profiled 98 mature microRNAs (miRNAs) using a stem-loop reverse transcription polymerase chain reaction assay array based on human miRNAs. We demonstrated that one, but not two, base-pair changes in the miRNA recognition sequence at the 3' end only marginally affected copy number estimates. Absolute levels of miRNAs were measured in matured cattle oocytes, eight-cell embryos and normal and parthenogenetic blastocysts and Day-14 trophoblast. Most miRNA concentrations were below the expected functional threshold required for effective repression of moderately to highly abundant target RNA. In oocytes and peri-embryonic genome activation embryos, miRNA 320, a member of the Dgcr8/Drosha-independent class of miRNAs, was expressed at greater than 1000 copies per embryo. miRNAs were more abundant at the eight-cell than the oocyte stage. miRNA concentrations per cell increased from the eight-cell to the blastocyst stage. Both the number of miRNA species and their expression levels were reduced in trophoblast tissue at Day 14. The parthenogenetic samples were more related in their miRNA expression profiles to each other than to their wild-type (in vitro-produced cultured) counterparts. miRNAs 299 and 323, which have been shown to be maternally expressed in other species, were also more than 4-fold overexpressed in the cattle parthenogenetic samples.

A mathematical model of the interaction between bovine blastocyst developmental stage and progesterone-stimulated uterine factors on differential embryonic development observed on Day 15 of gestation.

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.

Gene expression analysis of bovine embryonic disc, trophoblast and parietal hypoblast at the start of gastrulation.

In cattle early gastrulation-stage embryos (Stage 5), four tissues can be discerned: (i) the top layer of the embryonic disc consisting of embryonic ectoderm (EmE); (ii) the bottom layer of the disc consisting of mesoderm, endoderm and visceral hypoblast (MEH); (iii) the trophoblast (TB); and (iv) the parietal hypoblast. We performed microsurgery followed by RNA-seq to analyse the transcriptome of these four tissues as well as a developmentally earlier pre-gastrulation embryonic disc. The cattle EmE transcriptome was similar at Stages 4 and 5, characterised by the OCT4/SOX2/NANOG pluripotency network. Expression of genes associated with primordial germ cells suggest their presence in the EmE tissue at these stages. Anterior visceral hypoblast genes were transcribed in the Stage 4 disc, but no longer by Stage 5. The Stage 5 MEH layer was equally similar to mouse embryonic and extraembryonic visceral endoderm. Our data suggest that the first mesoderm to invaginate in cattle embryos is fated to become extraembryonic. TGFß, FGF, VEGF, PDGFA, IGF2, IHH and WNT signals and receptors were expressed, however the representative members of the FGF families differed from that seen in equivalent tissues of mouse embryos. The TB transcriptome was unique and differed significantly from that of mice. FGF signalling in the TB may be autocrine with both FGFR2 and FGF2 expressed. Our data revealed a range of potential inter-tissue interactions, highlighted significant differences in early development between mice and cattle and yielded insight into the developmental events occurring at the start of gastrulation.

The impact of ovarian stimulation protocol on oocyte quality, subsequent in vitro embryo development, and pregnancy after transfer to recipients in Eld's deer (Rucervus eldii thamin).

Propagating genetically valuable individuals through oocyte collection, in vitro fertilization (IVF) and embryo transfer is critical to maintain sustainable populations of the endangered Eld's deer. The objectives of this study were to assess the impact of exogenous FSH injections on (1) the number and in vitro competence of oocytes collected and (2) the developmental potential of resulting IVF embryos after transfer into recipients during the breeding season (February-April). In a pilot experiment, estrus synchronization was conducted in three surplus females (using intravaginal progesterone-releasing devices, CIDRG for 14 days and injections of buserelin (a GnRH agonist). Five days after CIDR removal, ovaries were excised, minced and a total of 133 oocytes were recovered. Following in vitro maturation (IVM) and IVF, 63% of the oocytes formed embryos but only 5% reached the blastocyst stage. In a subsequent study, follicle numbers and diameters were compared between synchronized does stimulated with 0 or 80 mg FSH (-FSH and +FSH; n = 8 does in each group) and oocytes collected either by laparoscopic ovum pick-up or ovariectomy. FSH stimulation increased the main follicular diameter from 2-3 mm to 4-5 mm (P < 0.05) but not the oocyte number (∼20/donor) or the percentage of good quality oocytes (57%) regardless of the treatment. FSH stimulation did not either affect the percentage of cleaved embryos after IVF (25-35%; P > 0.05). Lastly, embryos at the 2-to 8-cell stage (from either + FSH or -FSH groups) were transferred into the oviducts of 11 synchronized recipients. With the +FSH embryos, three pregnancies failed between 90 and 120 days of gestation and two fawns that were born preterm (Days 215 and 224 of gestation) died at birth. In the -FSH group one healthy female fawn was born on Day 234 of gestation. This is the first report of a successful in vitro embryo production and subsequent birth of a live Eld's deer fawn. Further investigations are required to improve IVM/IVF success and the developmental potential of the embryos.

Morphological and Gene Expression Changes in Cattle Embryos from Hatched Blastocyst to Early Gastrulation Stages after Transfer of In Vitro Produced Embryos.

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.

Sperm distribution and fertilization after unilateral and bilateral laparoscopic artificial insemination with frozen-thawed goat semen.

Generally, laparoscopic artificial insemination (LAI) provides a higher success rate than of cervical insemination in goats. However, the sperm distribution after LAI in goats remains unknown, particularly when frozen-thawed semen is used. This study evaluated the distribution of frozen-thawed goat spermatozoa after LAI and compared the effects of sperm numbers and deposition sites (unilateral and bilateral sites) on pregnancy rate. In experiment 1, the frozen-thawed spermatozoa were stained either with CellTracker Green CMFDA (CT-Green) or CellTracker Red CMPTX (CT-Red), and in vitro evaluations of viability and motility were performed. In experiment 2, the labeled spermatozoa were deposited via LAI into the left (CT-Green) and right (CT-Red) uterine horns (n = 4). After ovariohysterectomy (6 hours after insemination), the distributions of green- and red-colored spermatozoa were assessed via tissue section, flushing, and the oviductal contents were also collected. Experiment 3 was designed to test the pregnancy rates in a group of 120 does after LAI using different numbers of spermatozoa (60 and 120 × 10(6) sperm per LAI) and different deposition sites. The results demonstrated that the fluorochromes used in this study did not impair sperm motility or viability. Frozen-thawed goat spermatozoa can migrate transuterinally after LAI, as evidenced by the observations of both CT-Green- and CT-Red-labeled spermatozoa in both uterine horns. Lower numbers of spermatozoa (60 × 10(6)) that are inseminated unilaterally (either ipsilateral or contralateral to the site of ovulation) can efficiently be used for LAI in goats (with a 56.67% pregnancy rate).

Specific epiblast loss and hypoblast impairment in cattle embryos sensitized to survival signalling by ubiquitous overexpression of the proapoptotic gene BAD.

Early embryonic lethality is common, particularly in dairy cattle. We made cattle embryos more sensitive to environmental stressors by raising the threshold of embryo survival signaling required to overcome the deleterious effects of overexpressing the proapoptotic protein BAD. Two primary fibroblast cell lines expressing BAD and exhibiting increased sensitivity to stress-induced apoptosis were used to generate transgenic Day 13/14 BAD embryos. Transgenic embryos were normal in terms of retrieval rates, average embryo length or expression levels of the trophectoderm marker ASCL2. However both lines of BAD-tg embryos lost the embryonic disc and thus the entire epiblast lineage at significantly greater frequencies than either co-transferrred IVP controls or LacZ-tg embryos. Embryos without epiblast still contained the second ICM-derived lineage, the hypopblast, albeit frequently in an impaired state, as shown by reduced expression of the hypoblast markers GATA4 and FIBRONECTIN. This indicates a gradient of sensitivity (epiblast > hypoblast > TE) to BAD overexpression. We postulate that the greater sensitivity of specifically the epiblast lineage that we have seen in our transgenic model, reflects an inherent greater susceptibility of this lineage to environmental stress and may underlie the epiblast-specific death seen in phantom pregnancies.

Trophectoderm lineage determination in cattle.

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.

Nuclear transfer-specific defects are not apparent during the second week of embryogenesis in cattle.

Somatic cell nuclear transfer (NT)-specific effects on postblastocyst early cattle embryogenesis were investigated by comparison to in vitro-produced (IVP) embryos grown under identical conditions to embryonic days (E) 14 and 15. Recipient effects were excluded by transferring mixed batches of NT and IVP embryos into each cow. Embryo recovery rates, proportions with an epiblast and embryo, as well as epiblast dimensions did not differ between NT and IVP embryos. A developmental expression profile was determined for nine trophoblast markers, two inner cell mass (ICM)/epiblast markers, and E-cadherin at nine time points between E7 and E26, providing a molecular gene signature assay for developmental progression. Gene expression levels for these genes (Cdx2, Elf5, Mash2, Ifn-tau, Furin, Kunitz1, Pag11, Gata3, Oct4 and Ifitm3) were equal in NT and IVP embryos of equivalent length. Furthermore, the average residual deviation of all 10 genes did not differ significantly suggesting an overall "normality" in gene expression of E14/15 NT embryos. The absence of NT-specific defects during the second, highly selective, week of cattle embryogenesis is interpreted as supportive for the view that NT-associated defects are predominantly of an epigenetic nature.

Red deer cloned from antler stem cells and their differentiated progeny.

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.