Regulation of NANOG and SOX2 expression by activin A and a canonical WNT agonist in bovine embryonic stem cells and blastocysts

Bovine embryonic stem cells (ESC) have features associated with the primed pluripotent state including low expression of one of the core pluripotency transcription factors, NANOG. It has been reported that NANOG expression can be upregulated in porcine ESC by treatment with activin A and the WNT agonist CHIR99021. Accordingly, it was tested whether expression of NANOG and another pluripotency factor SOX2 could be stimulated by activin A and the WNT agonist CHIR99021. Immunoreactive NANOG and SOX2 were analyzed for bovine ESC lines derived under conditions in which activin A and CHIR99021 were added singly or in combination. Activin A enhanced NANOG expression but also reduced SOX2 expression. CHIR99021 depressed expression of both NANOG and SOX2. In a second experiment, activin A enhanced blastocyst development while CHIR99021 treatment impaired blastocyst formation and reduced number of blastomeres. Activin A treatment decreased blastomeres in the blastocyst that were positive for either NANOG or SOX2 but increased those that were CDX2+ and that were GATA6+ outside the inner cell mass. CHIR99021 reduced SOX2+ and NANOG+ blastomeres without affecting the number or percent of blastomeres that were CDX2+ and GATA6+. Results indicate activation of activin A signaling stimulates NANOG expression during self-renewal of bovine ESC but suppresses cells expressing pluripotency markers in the blastocyst and increases cells expressing CDX2. Actions of activin A to promote blastocyst development may involve its role in promoting trophectoderm formation. Furthermore, results demonstrate the negative role of canonical WNT signaling in cattle for pluripotency marker expression in ESC and in formation of the inner cell mass and epiblast during embryonic development. This article has an associated First Person interview with the first author of the paper

An improved method for specific-target preamplification PCR analysis of single blastocysts useful for embryo sexing and high-throughput gene expression analysis

Gene expression analysis in preimplantation embryos has been used for answering fundamental questions related to development, prediction of pregnancy outcome, and other topics. Limited amounts of mRNA in preimplantation embryos hinders progress in studying the preimplantation embryo. Here, a method was developed involving direct synthesis and specific-target preamplification (STA) of cDNA for gene expression analysis in single blastocysts. Effective cell lysis and genomic DNA removal steps were incorporated into the method. In addition, conditions for real-time PCR of cDNA generated from these processes were improved. By using this system, reliable embryo sexing results based on expression of sex-chromosome linked genes was demonstrated. Calibration curve analysis of PCR results using the Fluidigm Biomark microfluidic platform (Fluidigm, South San Francisco, CA) was performed to evaluate 96 STA cDNA from single blastocysts. In total, 93.75% of the genes were validated. Robust amplification was detected even when STA cDNA from a single blastocyst was diluted 1,024-fold. Further analysis showed that within-assay variation increased when cycle threshold values exceeded 18. Overall, STA quantitative real-time PCR analysis was shown to be useful for analysis of gene expression of multiple specific targets in single blastocysts

Transcriptional metabolic reprogramming implements meiotic fate decision in mouse testicular germ cells

Summary: Nutrient starvation drives yeast meiosis, whereas retinoic acid (RA) is required for mammalian meiosis through its germline target Stra8. Here, by using single-cell transcriptomic analysis of wild-type and Stra8-deficient juvenile mouse germ cells, our data show that the expression of nutrient transporter genes, including Slc7a5, Slc38a2, and Slc2a1, is downregulated in germ cells during meiotic initiation, and this process requires Stra8, which binds to these genes and induces their H3K27 deacetylation. Consequently, Stra8-deficient germ cells sustain glutamine and glucose uptake in response to RA and exhibit hyperactive mTORC1/protein kinase A (PKA) activities. Importantly, expression of Slc38a2, a glutamine importer, is negatively correlated with meiotic genes in the GTEx dataset, and Slc38a2 knockdown downregulates mTORC1/PKA activities and induces meiotic gene expression. Thus, our study indicates that RA via Stra8, a chordate morphogen pathway, induces meiosis partially by generating a conserved nutrient restriction signal in mammalian germ cells by downregulating their nutrient transporter expression

Supplementary tables for Effect of reproductive management programs that prioritized AI at detected estrus or timed AI on the reproductive performance of primiparous Holstein cows of different genetic merit for fertility

Our objective was to compare reproductive outcomes of primiparous lactating Holstein cows of different genetic merit for fertility submitted for insemination with management programs that prioritized AI at detected estrus (AIE) or timed AI (TAI). Moreover, we aimed to determine if subgroups of cows with different fertility potential would present a distinct response to the reproductive management strategies compared. Lactating primiparous Holstein cows (n = 6 commercial farms) were stratified into high (Hi-Fert), medium (Med-Fert), and low (Lo-Fert) genetic fertility groups (FG) based on a Reproduction Index (RI) value calculated from multiple genomically enhanced predicted transmitting abilities (gPTA). Within herd and fertility group, cows were randomly assigned either to a program that prioritized TAI and had an extended VWP (P-TAI; n = 1,338) or another that prioritized AIE (P-AIE; n = 1,416) and used TAI for cows not AIE. Cows in P-TAI received first service by TAI at 84 ± 3 DIM after a Double-Ovsynch protocol, were AIE if detected in estrus after a previous AI and received TAI after an Ovsynch-56 protocol at 35 ± 3 d after a previous AI if a corpus luteum (CL) was visualized at nonpregnancy diagnosis (NPD) 32 ± 3 d after AI. Cows with no CL visualized at NPD received TAI at 42 ± 3 d after AI after an Ovsynch-56 protocol with progesterone supplementation (P4-Ovsynch). Cows in P-AIE were eligible for AIE after a PGF2α treatment at 53 ± 3 DIM and after a previous AI. Cows not AIE by 74 ± 3 DIM or by NPD 32 ± 3 d after AI received P4-Ovsynch for TAI at 74 ± 3 DIM or 42 ± 3 d after AI. Binary data were analyzed with logistic regression, count data with Poisson regression, continuous data by ANOVA, and time to event data by Cox’s proportional hazard regression. Pregnancy per AI (P/AI) to first service was greater for cows in the Hi- (59.8%) than the Med- (53.6%) and Lo-Fert (47.7%) groups, and for the P-TAI (58.7%) than the P-AIE (48.7%) treatment. Overall, P/AI for all second and subsequent AI combined did not differ by treatment (P-TAI = 45.2%; P-AIE = 44.5%) or FG (Hi-Fert = 46.1%; Med-Fert = 46.0%; Lo-Fert = 42.4%). The hazard of pregnancy after calving was greater for the P-AIE than the P-TAI treatment [hazard ratio (HR) = 1.27, 95% CI: 1.17 to 1.37)], and for the Hi-Fert than the Med-Fert (HR = 1.16, 95% CI: 1.05 to 1.28) and Lo-Fert (HR = 1.34, 95% CI: 1.20 to 1.49) groups. More cows in the Hi- (91.2%) than the Med- (88.4%) and Lo-Fert (85.8%) groups were pregnant at 200 DIM. Within FG, the hazard of pregnancy was greater for the P-AIE than the P-TAI treatment for the Hi-Fert (HR = 1.41, 95% CI: 1.22 to 1.64) and Med-Fert (HR = 1.28, 95% CI: 1.12 to 1.46) groups but not for the Lo-Fert group (HR = 1.13, 95% CI: 0.98 to 1.31). We conclude that primiparous Holstein cows of superior genetic merit for fertility had better reproductive performance than cows of inferior genetic merit for fertility regardless of the type of reproductive management used. In addition, the effect of programs that prioritized AIE or TAI on reproductive performance for cows of superior or inferior genetic merit for fertility depended on the outcomes evaluated. Thus, programs that prioritize AIE or TAI could be used to affect certain outcomes of reproductive performance or management