Machine learning to identify endometrial biomarkers predictive of pregnancy success following artificial insemination in dairy cows.

The objective was to identify a set of genes whose transcript abundance is predictive of a cow's ability to become pregnant following artificial insemination (AI). Endometrial epithelial cells from the uterine body were collected for RNA sequencing using the cytobrush method from 193 first-service Holstein cows at estrus prior to AI (day 0). A group of 253 first-service cows not used for cytobrush collection were controls. There was no effect of cytobrush collection on pregnancy outcomes at day 30 or 70 or on pregnancy loss between day 30 and 70. There were 2 upregulated and 214 downregulated genes (FDR < 0.05, absolute fold change >2-fold) for cows pregnant at day 30 versus those that were not pregnant. Functional terms overrepresented in the downregulated genes included those related to immune and inflammatory responses. Machine learning for fertility biomarkers with the R package BORUTA resulted in identification of 57 biomarkers that predicted pregnancy outcome at day 30 with an average accuracy of 77%. Thus, machine learning can identify predictive biomarkers of pregnancy in endometrium with high accuracy. Moreover, sampling of endometrial epithelium using the cytobrush can help understand functional characteristics of the endometrium at AI without compromising cow fertility. Functional characteristics of the genes comprising the set of biomarkers is indicative that a major determinant of cow fertility, at least for first insemination after calving, is immune status of the uterus, which, in turn, is likely to reflect the previous history of uterine disease.

Meta-analysis to determine efficacy of colony-stimulating factor 2 for improving pregnancy success after embryo transfer in cattle.

Results have been inconsistent as to whether addition of colony stimulating factor 2 (CSF2) to culture medium improves embryo competence for establishment of pregnancy in cattle and humans. The purpose of the current study was to use all available experiments in cattle concerning effects of CSF2 on pregnancy success after transfer into recipient cattle. The approach was to perform a meta-analysis of all published data sets as well as data from an unpublished experiment described for the first time here. Meta-analysis failed to support the hypothesis that addition of CSF2 to embryo culture medium improves competence of bovine blastocysts to increase pregnancy or calving rates after transfer into recipient females. Thus, its general use as a culture medium additive to increase pregnancy success after embryo transfer is not recommended.

Identification of quantitative trait loci and associated candidate genes for pregnancy success in Angus-Brahman crossbred heifers.

BACKGROUND: In beef cattle, more than 50% of the energy input to produce a unit of beef is consumed by the female that produced the calf. Development of genomic tools to identify females with high genetic merit for reproductive function could increase the profitability and sustainability of beef production. RESULTS: Genome-wide association studies (GWAS) were performed using a single-step genomic best linear unbiased prediction approach on pregnancy outcome traits from a population of Angus-Brahman crossbred heifers. Furthermore, a validation GWAS was performed using data from another farm. Heifers were genotyped with the Bovine GGP F250 array that contains 221,077 SNPs. In the discovery population, heifers were bred in winter breeding seasons involving a single round of timed artificial insemination (AI) followed by natural mating for 3 months. Two phenotypes were analyzed: pregnancy outcome to first-service AI (PAI; n = 1,481) and pregnancy status at the end of the breeding season (PEBS; n = 1,725). The heritability was estimated as 0.149 and 0.122 for PAI and PEBS, respectively. In the PAI model, one quantitative trait locus (QTL), located between 52.3 and 52.5 Mb on BTA7, explained about 3% of the genetic variation, in a region containing a cluster of γ-protocadherin genes and SLC25A2. Other QTLs explaining between 0.5% and 1% of the genetic variation were found on BTA12 and 25. In the PEBS model, a large QTL on BTA7 was synonymous with the QTL for PAI, with minor QTLs located on BTA5, 9, 10, 11, 19, and 20. The validation population for pregnancy status at the end of the breeding season were Angus-Brahman crossbred heifers bred by natural mating. In concordance with the discovery population, the large QTL on BTA7 and QTLs on BTA10 and 12 were identified. CONCLUSIONS: In summary, QTLs and candidate SNPs identified were associated with pregnancy outcomes in beef heifers, including a large QTL associated with a group of protocadherin genes. Confirmation of these associations with larger populations could lead to the development of genomic predictions of reproductive function in beef cattle. Moreover, additional research is warranted to study the function of candidate genes associated with QTLs.

Transcriptomic profiling of the bovine endosalpinx and endometrium to identify putative embryokines.

The objectives of the present study were to characterize the expression of genes encoding for cell signaling ligands in the bovine endosalpinx and endometrium and analyze spatial changes in gene expression. RNA sequencing was performed for the endosalpinx from the ampulla of the oviduct and endometrium from the upper and middle uterine horn and uterine body at day 2 after ovulation from ipsilateral and contralateral sides relative to the ovulatory ovary. Of the 17,827 unique mRNA transcripts mapped, 2,072 were affected by cranial-caudal position in the reproductive tract and 818 were affected by side (false discovery rate < 0.05). There were 334 genes encoding for cell signaling ligands, with 128 genes having greater than two transcripts per million on average. A total of 81 cell signaling ligand genes were affected by position and 24 were affected by side. A data set of the transcriptome of two to four cell embryos was used to identify cell signaling ligand genes that were highly expressed in the ampulla for which there was high expression of the receptor in the embryo. The most expressed ligand-receptor pairs were PSAP/SORT1, MIF/CXCR4, GPI/AMFR, and KITLG/KIT. These cell signaling ligands, as well as others whose gene is expressed in the endosalpinx and endometrium, may influence early embryonic development. Spatial changes throughout the reproductive tract highlight the distinctive expression profile of the oviduct versus the endometrium, including a set of the identified genes encoding for cell signaling ligands, and highlight the local influence of the ovary. The results also show the continuity of expression for large numbers of genes in the reproductive tract.NEW & NOTEWORTHY Examination of the transcriptome of the endosalpinx and endometrium revealed the degree to which gene expression in the reproductive tract varies spatially. The expression of genes encoding cell signaling molecules that could potentially regulate embryonic development was also identified.

Review: Some challenges and unrealized opportunities toward widespread use of the in vitro-produced embryo in cattle production.

The embryo produced by in vitro oocyte maturation, fertilization, and embryonic development is an important resource for genetic improvement and has the potential to improve female fertility and to be programmed to produce offspring with superior ability for health and production. The cultured embryo is also an important component of several realized and potential technologies such as gene editing, somatic cell nuclear cloning, stem cell technologies and gamete generation in vitro. Full realization of the opportunities afforded by the in vitro-produced embryo will require overcoming some technical obstacles to cost-effective implementation of an embryo transfer program. Among the research goals for improving the penetration of embryo transfer in the cattle industry are development of methods to increase the supply of oocytes from genetically elite females, enhance the proportion of oocytes that become transferrable embryos, improve the fraction of embryos that establish pregnancy after transfer, reduce pregnancy wastage after pregnancy diagnosis, and identify culture conditions to optimize postnatal phenotype.

Presence of KREMEN receptors for DKK1 in the preimplantation bovine embryo.

The WNT inhibitory protein DKK1 has been shown to regulate development of the preimplantation embryo to the blastocyst stage. In cattle, DKK1 increases the number of trophectoderm cells that are the precursor of the placenta. DKK1 can affect cells by blocking WNT signaling through its receptors KREMEN1 and KREMEN2. Here it was shown that the mRNA for KREMEN1 and KREMEN2 decline as the embryo advances in development. Nonetheless, immunoreactive KREMEN1 was identified in blastocysts using Western blotting. DKK1 also decreased amount of immunoreactive CTNNB1 in blastocysts, as would be expected if DKK1 was signaling through a KREMEN-mediated pathway. Thus, it is likely that KREMEN1 functions as a receptor for DKK1 in the preimplantation bovine embryo.

Absence of a molecular circadian clock in the preimplantation embryo is a conserved characteristic in the mammal.

In brief: It is not known when a functional circadian clock is established in the developing embryo. Lack of expression of key genes involved in the clock mechanism is indicative that a functional circadian clock mechanism is absent in the mammalian preimplantation embryo through the blastocyst stage of development. Abstract: An embryonic circadian clock could conceivably organize cellular and developmental events temporally and in synchrony with other circadian rhythms in the mother. The hypothesis that a functional molecular clock exists in the preimplantation bovine, pig, human, and mouse embryo was tested by using publicly available RNAseq datasets to examine developmental changes in expression of the core genes responsible for the circadian clock - CLOCK, ARNTL, PER1, PER2, CRY1, and CRY2. In general, the transcript abundance of each gene decreased as development advanced to the blastocyst stage. The most notable exception was for CRY2, where transcript abundance was low and constant from the two-cell or four-cell to the blastocyst stage. Developmental patterns were generally the same for all species although there were some species-specific patterns such as an absence of PER1 expression in the pig, an increase in ARNTL expression at the four-cell stage in human, and an increase in expression of Clock and Per1 from the zygote to two-cell stage in the mouse. Analysis of intronic reads (indicative of embryonic transcription) for bovine embryos indicated an absence of embryonic transcription. Immunoreactive CRY1 was not detected in the bovine blastocyst. Results indicate that the preimplantation mammalian embryo lacks a functional intrinsic clock although specific components of the clock mechanism could conceivably play a role in other functions in the embryo.

Reflections on a career as graduate mentor-from baby steps at Wisconsin to today.

Graduate education is an important aspect of the life of most academic scientists and a serious responsibility because it comes with the obligation to help students achieve their career and life goals. It can also be very fulfilling for the graduate mentor in terms of personal satisfaction and advancement of the research program. Learning to be a good major professor is an active process that depends on developing a formal framework of education and modifying that framework for each student based on past experiences and experimentation, advice from colleagues, and the individual personality of the student. Perhaps most important is for the graduate mentor to buy into the success and well-being of the student. Among the characteristics that a major professor could seek to instill in his or her students are critical and independent thinking, self-confidence, a thick skin, teamwork, laboratory skills and understanding, and the ability for hard work. Work to make science joyful by celebrating accomplishments, creating a fun environment in the lab, and stressing the societal value of science as compared to personal rewards or ambition.

Science is dependent on education of the next generation through the formal and informal instruction that new scientists experience in graduate school. This paper focuses on some approaches to be considered by the graduate mentor as he or she guides his students into the world of science.

Disruption of CSF2RA in the bovine preimplantation embryo reduces development and affects embryonic gene expression in utero.

The hypothesis that CSF2 plays a role in the preimplantation development of the bovine embryo was tested by evaluating consequences of inactivation of CSF2RA (the functional receptor in the embryo) for development of embryos in utero. CRISPR/Cas9 was used to alter sequences on exon 5 and intron 5 of CSF2RA, Control embryos were injected with Cas9 mRNA only. Embryos > 16 cells at day 5 after insemination were transferred to synchronized recipient females in groups of 7 to 24. Embryos were flushed from the uterus two days later. The proportion of recovered embryos that developed to the blastocyst stage was lower for knockout embryos (39%) than for control embryos (63%). RNA sequencing of individual morulae and blastocysts indicated a total of 27 (morula) or 15 (blastocyst) differentially-expressed genes (false discovery rate <0.05). Gene set enrichment analysis indicated that the knockout affected genes playing roles in several functions including cell signaling and glycosylation. It was concluded that signaling through CSF2RA is not obligatory for development of the bovine preimplantation embryo to the blastocyst stage but that CSF2 signaling does enhance the likelihood that the embryo can become a blastocyst and result in specific changes in gene expression.

Machine-learning methods applied to integrated transcriptomic data from bovine blastocysts and elongating conceptuses to identify genes predictive of embryonic competence.

Early pregnancy loss markedly impacts reproductive efficiency in cattle. The objectives were to model a biologically relevant gene signature predicting embryonic competence for survival after integrating transcriptomic data from blastocysts and elongating conceptuses with different developmental capacities and to validate the potential biomarkers with independent embryonic data sets through the application of machine-learning algorithms. First, two data sets from in vivo-produced blastocysts competent or not to sustain a pregnancy were integrated with a data set from long and short day-15 conceptuses. A statistical contrast determined differentially expressed genes (DEG) increasing in expression from a competent blastocyst to a long conceptus and vice versa; these were enriched for KEGG pathways related to glycolysis/gluconeogenesis and RNA processing, respectively. Next, the most discriminative DEG between blastocysts that resulted or did not in pregnancy were selected by linear discriminant analysis. These eight putative biomarker genes were validated by modeling their expression in competent or noncompetent blastocysts through Bayesian logistic regression or neural networks and predicting embryo developmental fate in four external data sets consisting of in vitro-produced blastocysts (i) competent or not, or (ii) exposed or not to detrimental conditions during culture, and elongated conceptuses (iii) of different length, or (iv) developed in the uteri of high- or subfertile heifers. Predictions for each data set were more than 85% accurate, suggesting that these genes play a key role in embryo development and pregnancy establishment. In conclusion, this study integrated transcriptomic data from seven independent experiments to identify a small set of genes capable of predicting embryonic competence for survival.

Colony stimulating factor 2 protects the preimplantation bovine embryo from heat shock.

Heat stress can have severe deleterious effects on embryo development and survival. The present study evaluated whether CSF2 can protect the developmental competence of the bovine embryo following exposure to a heat shock of 41°C at the zygote and morula stages. In the first experiment, putative zygotes and 2-cell embryos were assigned to receive either 10 ng/ml CSF2 or vehicle, and then cultured for 15 h at either 38.5°C or 41°C and then at 38.5°C until day 7.5. Heat shock reduced blastocyst development for embryos treated with vehicle but not for embryos cultured with CSF2. In the second experiment, day 5 embryos (morula) were treated with CSF2 or vehicle and then cultured for 15 h at either 38.5°C or 41°C and then at 38.5°C until day 7.5. Temperature treatment did not affect development to the blastocyst stage and there was no effect of CSF2 treatment or the interaction. Results indicate that CSF2 can reduce the deleterious effects of heat shock at the zygote or two-cell stage when the embryo is transcriptionally inactive.

Physiological responses of Holstein calves and heifers carrying the SLICK1 allele to heat stress in California and Florida dairy farms.

Inheritance of the SLICK1 allele of the prolactin receptor gene improves thermotolerance of lactating Holstein cows under humid heat stress conditions. The aim of this study was to investigate whether pre- and postweaning Holstein heifers carrying the SLICK1 allele would show physiological responses indicative of higher tolerance to heat stress in high- and low-humidity climates. A total of 101 heifer calves of two age groups heterozygous for the SLICK1 allele and 103 wild-type half-siblings were evaluated during July 2020 in 3 dairy farms in central California and 2 in south Florida. Dry bulb temperature and relative humidity data were recorded during evaluation and used to calculate the temperature-humidity index (THI). Physiological measurements were obtained between 1600 and 1900 h in California, and 1200 and 1400 h in Florida and included rectal temperature, respiration rate, skin temperature, and sweating rate. Data were analyzed via Generalized Linear Mixed Models including the main effects of genotype, state, group, sire, farm within state, and interactions, with THI included as a covariate. The correlations between THI and dependent variables were analyzed via linear regression. The average 24-h THI was higher in Florida compared with California (90 vs. 72, respectively); the main driver of the higher THI in Florida was the high relative humidity (average 85.6% in Florida vs. 36.7% in California). In Florida, the rectal temperature of slick calves was 0.4°C lower than non-slick calves (39.5 ± 0.1 vs 39.9 ± 0.1°C); no differences were detected between slick and non-slick calves in California. Regardless of genotype, heifer calves in Florida had higher respiration rate, higher rectal and skin temperatures, and lower sweating rate than in California. This study is the first to evaluate physiological responses of calves carrying the SLICK1 allele under heat stress conditions in different climates. Our findings demonstrate that the presence of this allele is associated with lower rectal temperatures in pre- and post-weaning Holstein females. According to the physiological parameters evaluated, calves raised in Florida appeared to be under more severe heat stress; in those conditions, the SLICK1 allele was advantageous to confer thermotolerance as evidenced by lower rectal temperature in slick animals.

Effect of addition of ascorbate, dithiothreitol or a caspase-3 inhibitor to cryopreservation medium on post-thaw survival of bovine embryos produced in vitro.

Experiments were conducted to investigate whether supplementation of cryopreservation medium with ascorbate, dithiothreitol (DTT) or an inhibitor of caspase-3 (z-DEVD-fmk) could improve post-thaw survival of bovine embryos produced in vitro (IVP). For all experiments, embryos were harvested on day 7 after insemination and subjected to controlled-rate freezing in medium containing 1.5 M ethylene glycol and treatments as described below. In experiments 1-3, embryos were cryopreserved in freezing medium with ascorbate (0, 0.1, 0.3 or 0.5 mM), DTT (0, 50, 100 or 200 µM) and z-DEVD-fmk (0, 50, 100 or 200 µM), respectively. Post-thaw survival was assessed at 24, 48 and 72 h. For experiments 4-5, embryos were cryopreserved in freezing medium with or without 0.1 mM ascorbate. At 24 h post-thaw, embryo total cell number, DNA fragmentation and levels of reactive oxygen species (ROS) were evaluated. Embryos subjected to freezing and thawing in medium supplemented with 0.1 mM ascorbate had greater (p < .05) re-expansion rates at 24, 48 and 72 h and hatching rate at 72 h as compared to embryos not treated with ascorbate. Post-thaw cryosurvival was not affected by the addition of either DTT or z-DEVD-fmk to medium used for cryopreservation. Embryos cryopreserved in medium supplemented with 0.1 mM ascorbate had reduced (p < .001) levels of intracellular ROS and fewer (p < .001) cells with DNA fragmentation. In conclusion, post-thaw survival of bovine IVP embryos is enhanced by supplementation of freezing medium with ascorbate.

Actions of DKK1 on the preimplantation bovine embryo to affect pregnancy establishment, placental function, and postnatal phenotype†.

One mechanism by which the maternal environment regulates the early embryo is by secretion of cell-signaling molecules. One of these is dickkopf WNT signaling pathway inhibitor 1. Objectives were to (A) resolve discrepancies in the literature regarding effects of dickkopf WNT signaling pathway inhibitor 1 in the bovine embryo on development of trophectoderm and competence to establish pregnancy after embryo transfer and (B) determine whether there are long-term consequences of dickkopf WNT signaling pathway inhibitor 1 on placental function and postnatal phenotype. Embryos produced in vitro were cultured with vehicle or 100 ng/mL recombinant human dickkopf WNT signaling pathway inhibitor 1 from Days 5 to 7.5 of development (i.e., the morula and blastocyst stages of development). dickkopf WNT signaling pathway inhibitor 1 increased the number of cells positive for the trophectoderm marker CDX2 at Day 7.5 of development while having no effect on number of cells positive for the inner cell mass marker SOX2. There was no effect of dickkopf WNT signaling pathway inhibitor 1 on pregnancy or calving rate after transfer of blastocysts produced with Y-sorted semen to either lactating dairy cows or suckling beef cows. Treatment with dickkopf WNT signaling pathway inhibitor 1 at the morula-to-blastocyst stages programmed placental function, as measured by an effect of dickkopf WNT signaling pathway inhibitor 1 on plasma concentrations of pregnancy associated glycoproteins and placental lactogen at Day 160 of gestation (although not on other days examined). dickkopf WNT signaling pathway inhibitor 1 treatment also resulted in calves that were heavier at birth as compared to calves derived from control embryos. After birth, dickkopf WNT signaling pathway inhibitor 1 calves grew slower than controls. Results confirm that dickkopf WNT signaling pathway inhibitor 1 alters the developmental program of the bovine embryo to affect both prenatal and postnatal phenotypes.

Actions of WNT family member 5A to regulate characteristics of development of the bovine preimplantation embryo†.

WNT signaling is important for regulation of embryonic development. The most abundant WNT gene expressed in the bovine endometrium during the preimplantation period is WNT5A. One objective was to determine whether WNT5A regulates competence of the bovine preimplantation embryo to become a blastocyst and alters the number of cells in the inner cell mass and trophectoderm. A second objective was to delineate features of the cell-signaling mechanisms involved in WNT5A actions. WNT5A caused a concentration-dependent increase in the proportion of embryos developing to the blastocyst stage and in the number of inner cell mass cells in the resultant blastocysts. A concentration of 200 ng/mL was most effective, and a higher concentration of 400 ng/mL was not stimulatory. Bovine serum albumin in culture reduced the magnitude of effects of WNT5A on development to the blastocyst stage. WNT5A affected expression of 173 genes at the morula stage; all were upregulated by WNT5A. Many of the upregulated genes were associated with cell signaling. Actions of WNT5A on development to the blastocyst stage were suppressed by a Rho-associated coiled-coil kinase (ROCK) signaling inhibitor, suggesting that WNT5A acts through Ras homology gene family member A (RhoA)/ROCK signaling. Other experiments indicated that actions of WNT5A are independent of the canonical ß-catenin signaling pathway and RAC1/c-Jun N-terminal kinase (JNK) signaling. This is the first report outlining the actions of WNT5A to alter the development of the mammalian embryo. These findings provide insights into how embryokines regulate maternal-embryonic communication.

Actions of CSF2 and DKK1 on bovine embryo development and pregnancy outcomes are affected by composition of embryo culture medium.

Procedures for in vitro embryo production in cattle have not been optimized. In the current experiment, we utilized a 3 × 3 factorial design to test whether the proportion of embryos becoming blastocysts in culture and the pregnancy rate after embryo transfer are affected by type of serum in the medium [no serum; 3% (v/v) KnockOut Serum Replacement (SR); 3% (v/v) fetal bovine serum (FBS)] and addition of specific embryokines [vehicle; 10 ng/mL colony stimulating factor 2 (CSF2); 100 ng/mL dickkopf related protein 1 (DKK1)] at day 5 of culture. Embryos were produced using abattoir-derived ovaries and Y-sorted semen from two Angus sires. The percent of putative zygotes and cleaved embryos becoming blastocysts was improved by SR and FBS. Pregnancy rate at day 30 was determined for 1426 Nelore recipients and calving rate for 266 recipients. In the absence of CSF2 or DKK1, pregnancy rates were lower for embryos cultured with SR or FBS. CSF2 and DKK1 reduced pregnancy rate for embryos cultured without serum but had no detrimental effect in the SR or FBS groups. Indeed, CSF2 blocked the negative effect of FBS on pregnancy rate. Data on birth weights were available for 67 bull calves. There were no effects of treatment. The sire used to produce embryos had significant and large effects on development to the blastocyst stage, pregnancy rate at day 30, calving rate and pregnancy loss between day 30 and calving. Results indicate that (1) SR and FBS can improve embryonic development in vitro while also compromising competence of embryos to survive after transfer, (2) actions of CSF2 and DKK1 depend upon other characteristics of the embryo production system, and (3) sire can have a large effect on embryonic development before and after transfer.

Effects of the SLICK1 mutation in PRLR on regulation of core body temperature and global gene expression in liver in cattle.

The SLICK1 mutation in bovine PRLR (c.1382del; rs517047387) is a deletion mutation resulting in a protein with a truncated intracellular domain. Cattle carrying at least one allele have a phenotype characterized by a short hair coat (slick phenotype) and increased resistance to heat stress. Given the pleiotropic nature of prolactin, the mutation may affect other physiological characteristics. The liver is one organ that could potentially be affected because of the expression of PRLR. The mutation is a dominant allele, and heterozygous animals have a similar hair coat to that of animals homozygous for the mutation. Present objectives were to determine whether inheritance of the SLICK1 mutation affects liver gene expression and if animals homozygous for the SLICK1 allele differ from heterozygotes in liver gene expression and regulation of body temperature during heat stress. In one experiment, rectal and ruminal temperatures were less for Holstein heifers that were heterozygous for the SLICK1 allele compared with wildtype heifers. There were 71 differentially expressed genes in liver, with 13 upregulated and 58 downregulated in SLICK1 heterozygotes. Among the ontologies characteristic of differentially expressed genes were those related to immune function and fatty acid and amino acid metabolism. In a prospective cohort study conducted with adult Senepol cattle, body temperature and hepatic gene expression were compared between animals heterozygous or homozygous for the SLICK1 mutation. There were no differences in ruminal temperatures between genotypes, rectal temperature was higher in animals homozygous for the SLICK1 mutation, and there was only one gene in liver that was differentially expressed. It was concluded that inheritance of the SLICK1 allele can exert functional changes beyond those related to hair growth although changes in liver gene expression were not extensive. Results are also consistent with the SLICK1 allele being dominant because there were few differences in phenotype between animals inheriting one or two copies of the allele.

RhoA/ROCK signaling antagonizes bovine trophoblast stem cell self-renewal and regulates preimplantation embryo size and differentiation.

Exponential proliferation of trophoblast stem cells (TSC) is crucial in Ruminantia to maximize numerical access to caruncles, the restricted uterine sites that permit implantation. When translating systems biology of the undifferentiated bovine trophectoderm, we uncovered that inhibition of RhoA/Rock promoted self-renewing proliferation and substantially increased blastocyst size. Analysis of transcripts suppressed by Rock inhibition revealed transforming growth factor ß1 (TGFß1) as a primary upstream effector. TGFß1 treatment induced changes consistent with differentiation in bTSCs, a response that could be replicated by induced expression of the bovine ROCK2 transgene. Rocki could partially antagonize TGFß1 effects, and TGFß receptor inhibition promoted proliferation identical to Rocki, indicating an all-encompassing upstream regulation. Morphological differentiation included formation of binucleate cells and infrequent multinucleate syncytia, features we also localize in the in vivo bovine placenta. Collectively, we demonstrate a central role for TGFß1, RhoA and Rock in inducing bTSC differentiation, attenuation of which is sufficient to sustain self-renewal and proliferation linked to blastocyst size and preimplantation development. Unraveling these mechanisms augments evolutionary/comparative physiology of the trophoblast cell lineage and placental development in eutherians.

Future of biomedical, agricultural, and biological systems research using domesticated animals.

Increased knowledge of reproduction and health of domesticated animals is integral to sustain and improve global competitiveness of U.S. animal agriculture, understand and resolve complex animal and human diseases, and advance fundamental research in sciences that are critical to understanding mechanisms of action and identifying future targets for interventions. Historically, federal and state budgets have dwindled and funding for the United States Department of Agriculture (USDA) National Institute of Food and Agriculture (NIFA) competitive grants programs remained relatively stagnant from 1985 through 2010. This shortage in critical financial support for basic and applied research, coupled with the underappreciated knowledge of the utility of non-rodent species for biomedical research, hindered funding opportunities for research involving livestock and limited improvements in both animal agriculture and animal and human health. In 2010, the National Institutes of Health and USDA NIFA established an interagency partnership to promote the use of agriculturally important animal species in basic and translational research relevant to both biomedicine and agriculture. This interagency program supported 61 grants totaling over $107 million with 23 awards to new or early-stage investigators. This article will review the success of the 9-year Dual Purpose effort and highlight opportunities for utilizing domesticated agricultural animals in research.

Progesterone-dependent and progesterone-independent modulation of luminal epithelial transcription to support pregnancy in cattle.

In cattle, starting 4-5 days after estrus, preimplantation embryonic development occurs in the confinement of the uterine lumen. Cells in the endometrial epithelial layer control the molecular traffic to and from the lumen and, thereby determine luminal composition. Starting early postestrus, endometrial function is regulated by sex steroids, but the effects of progesterone on luminal cells transcription have not been measured in vivo. The first objective was to determine the extent to which progesterone controls transcription in luminal epithelial cells 4 days (D4) after estrus. The second objective was to discover luminal transcripts that predict pregnancy outcomes when the effect of progesterone is controlled. Endometrial luminal epithelial cells were collected from embryo transfer recipients on D4 using a cytological brush and their transcriptome was determined by RNASeq. Pregnancy by embryo transfer was measured on D30 (25 pregnant and 18 nonpregnant). Progesterone concentration on D4 was associated positively (n = 182) and negatively (n = 58) with gene expression. Progesterone-modulated transcription indicated an increase in oxidative phosphorylation, biosynthetic activity, and proliferation of epithelial cells. When these effects of progesterone were controlled, different genes affected positively (n = 22) and negatively (n = 292) odds of pregnancy. These set of genes indicated that a receptive uterine environment was characterized by the inhibition of phosphoinositide signaling and innate immune system responses. A panel of 25 genes predicted the pregnancy outcome with sensitivity and specificity ranging from 64%-96% and 44%-83%, respectively. In conclusion, in the early diestrus, both progesterone-dependent and progesterone-independent mechanisms regulate luminal epithelial transcription associated with pregnancy outcomes in cattle.