Embryonic microRNAs are essential for bovine preimplantation embryo development.

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression after transcription. miRNAs are present in transcriptionally quiescent full-grown oocytes and preimplantation embryos that display a low level of transcription prior to embryonic genome activation. The role of miRNAs, if any, in preimplantation development is not known. The temporal pattern of expression of miRNAs during bovine preimplantation development was determined by small RNA-sequencing using eggs and preimplantation embryos (1-cell, 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst). Embryos cultured in the presence of α-amanitin, which permitted the distinguishing of maternal miRNAs from embryonic miRNAs, indicated that embryonic miRNA expression was first detected at the two-cell stage but dramatically increased during the morula and blastocyst stages. Targeting DGCR8 by a small-interfering RNA/morpholino approach revealed a role for miRNAs in the morula-to-blastocyst transition. Knockdown of DGCR8 not only inhibited expression of embryonically expressed miRNAs but also inhibited the morula-to-blastocyst transition. In addition, RNA-sequencing identified an increased relative abundance of messenger RNAs potentially targeted by embryonic miRNAs in DGCR8-knockdown embryos when compared with controls. Results from these experiments implicate an essential role for miRNAs in bovine preimplantation embryo development.

Endometrial receptivity and embryo implantation in carnivores-commonalities and differences with other mammalian species.

Endometrial receptivity and embryo implantation processes are a major point of pregnancy failure in many mammalian species, including humans. Although reproductive biology in many carnivore species remains enigmatic, the few that have been studied so far are invaluable comparative models. The goals of this review are to (1) summarize current data on the mechanisms involved in uterine receptivity and embryo implantation in carnivores, including commonalities and differences with other mammalian species and (2) identify research priorities to better understand a key phenomenon in a critical group of mammals. Besides unique reproductive traits in some carnivores (induced vs. spontaneous ovulation in cats, ovulation at the germinal vesicle stage in dogs), preimplantation embryo development is comparable with other orders. However, the timing of implantation varies, especially in species having an embryonic diapause. Mechanisms involved in endometrial receptivity and decidualization still remain to be fully understood, but specific markers have already been identified. Importantly, the use of endogenous hormones to control the ovarian activity may impact endometrial receptivity and subsequent embryo implantation. Next, research efforts should take advantage of advanced technologies to further study embryo implantation in carnivores and to provide more relevant models to reproductive medicine or for the conservation of rare and endangered species.

Transcript profiling of bovine embryos implicates specific transcription factors in the maternal-to-embryo transition.

Full-grown oocytes are transcriptionally quiescent. Following maturation and fertilization, the early stages of embryonic development occur in the absence (or low levels) of transcription that results in a period of development relying on maternally derived products (e.g., mRNAs and proteins). Two critical steps occur during the transition from maternal to embryo control of development: maternal mRNA clearance and embryonic genome activation with an associated dramatic reprogramming of gene expression required for further development. By combining an RNA polymerase II inhibitor with RNA sequencing, we were able not only to distinguish maternally derived from embryonic transcripts in bovine preimplantation embryos but also to establish that embryonic gene activation is required for clearance of maternal mRNAs as well as to identify putative transcription factors that are likely critical for early bovine development.

Derivation of sheep embryonic stem cells under optimized conditions.

Until recently, it has been difficult to derive and maintain stable embryonic stem cells lines from livestock species. Sheep ESCs with characteristics similar to those described for rodents and primates have not been produced. We report the derivation of sheep ESCs under a chemically defined culture system containing fibroblast growth factor 2 (FGF2) and a tankyrase/Wnt inhibitor (IWR1). We also show that several culture conditions used for stabilizing naïve and intermediate pluripotency states in humans and mice were unsuitable to maintain ovine pluripotency in vitro. Sheep ESCs display a smooth dome-shaped colony morphology, and maintain an euploid karyotype and stable expression of pluripotency markers after more than 40 passages. We further demonstrate that IWR1 and FGF2 are essential for the maintenance of an undifferentiated state in de novo derived sheep ESCs. The derivation of stable pluripotent cell lines from sheep blastocysts represents a step forward toward understanding pluripotency regulation in livestock species and developing novel biomedical and agricultural applications.

Adaptative response to changes in pyruvate metabolism on the epigenetic landscapes and transcriptomics of bovine embryos.

The epigenetic reprogramming that occurs during the earliest stages of embryonic development has been described as crucial for the initial events of cell specification and differentiation. Recently, the metabolic status of the embryo has gained attention as one of the main factors coordinating epigenetic events. In this work, we investigate the link between pyruvate metabolism and epigenetic regulation by culturing bovine embryos from day 5 in the presence of dichloroacetate (DCA), a pyruvate analog that increases the pyruvate to acetyl-CoA conversion, and iodoacetate (IA), which inhibits the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), leading to glycolysis inhibition. After 8 h of incubation, both DCA and IA-derived embryos presented higher mitochondrial membrane potential. Nevertheless, in both cases, lower levels of acetyl-CoA, ATP-citrate lyase and mitochondrial membrane potential were found in blastocysts, suggesting an adaptative metabolic response, especially in the DCA group. The metabolic alteration found in blastocysts led to changes in the global pattern of H3K9 and H3K27 acetylation and H3K27 trimethylation. Transcriptome analysis revealed that such alterations resulted in molecular differences mainly associated to metabolic processes, establishment of epigenetic marks, control of gene expression and cell cycle. The latter was further confirmed by the alteration of total cell number and cell differentiation in both groups when compared to the control. These results corroborate previous evidence of the relationship between the energy metabolism and the epigenetic reprogramming in preimplantation bovine embryos, reinforcing that the culture system is decisive for precise epigenetic reprogramming, with consequences for the molecular control and differentiation of cells.

Paternal genome rescues mouse preimplantation embryo development in the absence of maternally-recruited EZH2 activity.

Enhancer of zeste homolog 2 (EZH2), a component of the PRC2 complex, trimethylates H3K27, a transcriptionally repressive histone mark. EZH2 is encoded by a dormant maternal mRNA and inhibiting the maturation-associated increase in EZH2 activity using either a combined siRNA/morpholino approach or a small molecule inhibitor (GSK343) inhibits development of diploidized parthenotes to the blastocyst stage but not inseminated eggs, with longer GSK343 treatments leading to progressively greater inhibition of development. GSK343 treatment also results in a decrease in H3K27me3 and a decrease in global transcription in 2-cell parthenotes but not 2-cell embryos derived from inseminated eggs. RNA-sequencing revealed the relative abundance of ~100 zygotically-expressed transcripts is decreased by GSK treatment in parthenotes, but not in embryos, with many of the affected transcripts encoding proteins involved in transcription. A previous study found that parthenotes deficient in maternal Ezh2 readily develop to the blastocyst stage. To reconcile these differences we propose that the H3K27me3 state present in the zygote needs to be faithfully propagated following DNA replication in at least one pronucleus, otherwise development is compromised.

Can highly sensitive antimüllerian hormone testing predict failed response to ovarian stimulation?

OBJECTIVE: To determine whether a newer commercially available antimüllerian hormone (AMH) enzyme-linked immunosorbent assay (picoAMH ELISA, AnshLabs) with a lower threshold of detection is predictive of successful ovarian stimulation in a population of women with diminished ovarian reserve (DOR). DESIGN: Retrospective case-control study. SETTING: University-based IVF program. PATIENT(S): Cases were patients whose first IVF cycle was cancelled for lack of ovarian response (<3 follicles; n = 24). Controls were patients with DOR (early follicular FSH of ≥10 IU/L), whose first cycle resulted in aspiration of at least 3 oocytes (n = 24). INTERVENTION(S): Frozen serum samples collected during routine clinical care between 2008 and 2012 before starting IVF were analyzed for AMH using the picoAMH ELISA. MAIN OUTCOME MEASURE(S): Serum AMH levels in patients who successfully reached oocyte retrieval compared with patients with a failed controlled ovarian hyperstimulation (COH) cycle. Receiver operator curve analysis was used to identify a predictive threshold AMH value. RESULT(S): No demographic differences were found between groups. The successful group had a higher antral follicle count (8.5 vs. 6) and higher AMH levels (847 vs. 406 pg/mL). The AMH level correlated with the antral follicle count (R = 0.61). The AMH level of >500 pg/mL had 83.3% sensitivity and 70.8% specificity to detect patients who proceeded to successful oocyte retrieval. Below AMH levels of 100 pg/mL, no patients achieved oocyte retrieval. CONCLUSION(S): Due to a lower threshold of detection, picoAMH may be able to predict successful ovarian stimulation among women with DOR using a threshold of 500 pg/mL, with good sensitivity and specificity.