Chemical simulation of hypoxia in donor cells improves development of somatic cell nuclear transfer-derived embryos and increases abundance of transcripts related to glycolysis.

To improve efficiency of somatic cell nuclear transfer (SCNT), it is necessary to modify differentiated donor cells to become more amendable for reprogramming by the oocyte cytoplasm. A key feature that distinguishes somatic/differentiated cells from embryonic/undifferentiated cells is cellular metabolism, with somatic cells using oxidative phosphorylation (OXPHOS) while embryonic cells utilize glycolysis. Inducing metabolic reprogramming in donor cells could improve SCNT efficiency by priming cells to become more embryonic in nature before SCNT hypoxia inducible factor 1-α (HIF1-α), a transcription factor that allows for cell survival in low oxygen, promotes a metabolic switch from OXPHOS to glycolysis. We hypothesized that chemically stabilizing HIF1-α in donor cells by use of the hypoxia mimetic, cobalt chloride (CoCl2 ), would promote this metabolic switch in donor cells and subsequently improve the development of SCNT embryos. Donor cell treatment with 100 µM CoCl2 for 24 hr preceding SCNT upregulated messenfer RNA abundance of glycolytic enzymes, improved SCNT development to the blastocyst stage and quality, and affected gene expression in the blastocysts. After transferring blastocysts created from CoCl2 -treated donor cells to surrogates, healthy cloned piglets were produced. Therefore, shifting metabolism toward glycolysis in donor cells by CoCl2 treatment is a simple, economical way of improving the in vitro efficiency of SCNT and is capable of producing live animals.

Removal of hypotaurine from porcine embryo culture medium does not impair development of in vitro-fertilized or somatic cell nuclear transfer-derived embryos at low oxygen tension.

Hypotaurine (HT) is a routine component of porcine embryo culture medium, functioning as an antioxidant, but its requirement may be diminished as most embryo culture systems now use 5% O2 instead of atmospheric (20%) O2 . Our objective was to determine the effects of removing HT from the culture medium on porcine preimplantation embryo development. Embryos cultured in 20% O2 without HT had decreased blastocyst development compared to culture with HT or in 5% O2 with or without HT. Notably, differences in blastocyst development or total cell number were not detected between embryos cultured in 5% O2 with or without HT. After culture in 5% O2 without HT and embryo transfer, healthy fetuses were retrieved from two pregnancies on Day 42, confirming in vivo developmental competence. Transcript abundance of proapoptotic markers was decreased in embryos cultured without HT regardless of oxygen tension; however, assays for apoptosis did not demonstrate differences between groups. Additionally, no differences were observed in the development or apoptosis of somatic cell nuclear transfer-derived embryos cultured in 5% O2 with or without HT. With decreased utility in 5% O2 , removing HT from porcine embryo culture medium would also have economic advantages because it is undoubtedly the most expensive component.

Impact of Chorionic Somatomammotropin In Vivo RNA Interference Phenotype on Uteroplacental Expression of the IGF Axis.

While fetal growth is dependent on many factors, optimal placental function is a prerequisite for a normal pregnancy outcome. The majority of fetal growth-restricted (FGR) pregnancies result from placental insufficiency (PI). The insulin-like growth factors (IGF1 and IGF2) stimulate fetal growth and placental development and function. Previously, we demonstrated that in vivo RNA interference (RNAi) of the placental hormone, chorionic somatomammotropin (CSH), resulted in two phenotypes. One phenotype exhibits significant placental and fetal growth restriction (PI-FGR), impaired placental nutrient transport, and significant reductions in umbilical insulin and IGF1. The other phenotype does not exhibit statistically significant changes in placental or fetal growth (non-FGR). It was our objective to further characterize these two phenotypes by determining the impact of CSH RNAi on the placental (maternal caruncle and fetal cotyledon) expression of the IGF axis. The trophectoderm of hatched blastocysts (9 days of gestation, dGA) were infected with a lentivirus expressing either a non-targeting sequence (NTS RNAi) control or CSH-specific shRNA (CSH RNAi) prior to embryo transfer into synchronized recipient ewes. At ≈125 dGA, pregnancies were fitted with vascular catheters to undergo steady-state metabolic studies. Nutrient uptakes were determined, and tissues were harvested at necropsy. In both CSH RNAi non-FGR and PI-FGR pregnancies, uterine blood flow was significantly reduced (p ≤ 0.05), while umbilical blood flow (p ≤ 0.01), both uterine and umbilical glucose and oxygen uptakes (p ≤ 0.05), and umbilical concentrations of insulin and IGF1 (p ≤ 0.05) were reduced in CSH RNAi PI-FGR pregnancies. Fetal cotyledon IGF1 mRNA concentration was reduced (p ≤ 0.05) in CSH RNAi PI-FGR pregnancies, whereas neither IGF1 nor IGF2 mRNA concentrations were impacted in the maternal caruncles, and either placental tissue in the non-FGR pregnancies. Fetal cotyledon IGF1R and IGF2R mRNA concentrations were not impacted for either phenotype, yet IGF2R was increased (p ≤ 0.01) in the maternal caruncles of CSH RNAi PI-FGR pregnancies. For the IGF binding proteins (IGFBP1, IGFBP2, IGFBP3), only IGFBP2 mRNA concentrations were impacted, with elevated IGFBP2 mRNA in both the fetal cotyledon (p ≤ 0.01) and maternal caruncle (p = 0.08) of CSH RNAi non-FGR pregnancies. These data support the importance of IGF1 in placental growth and function but may also implicate IGFBP2 in salvaging placental growth in non-FGR pregnancies.

In vivo investigation of ruminant placenta function and physiology-a review.

The placenta facilitates the transport of nutrients to the fetus, removal of waste products from the fetus, immune protection of the fetus and functions as an endocrine organ, thereby determining the environment for fetal growth and development. Additionally, the placenta is a highly metabolic organ in itself, utilizing a majority of the oxygen and glucose derived from maternal circulation. Consequently, optimal placental function is required for the offspring to reach its genetic potential in utero. Among ruminants, pregnant sheep have been used extensively for investigating pregnancy physiology, in part due to the ability to place indwelling catheters within both maternal and fetal vessels, allowing for steady-state investigation of blood flow, nutrient uptakes and utilization, and hormone secretion, under non-stressed and non-anesthetized conditions. This methodology has been applied to both normal and compromised pregnancies. As such, our understanding of the in vivo physiology of pregnancy in sheep is unrivalled by any other species. However, until recently, a significant deficit existed in determining the specific function or significance of individual genes expressed by the placenta in ruminants. To that end, we developed and have been using in vivo RNA interference (RNAi) within the sheep placenta to examine the function and relative importance of genes involved in conceptus development (PRR15 and LIN28), placental nutrient transport (SLC2A1 and SLC2A3), and placenta-derived hormones (CSH). A lentiviral vector is used to generate virus that is stably integrated into the infected cell's genome, thereby expressing a short-hairpin RNA (shRNA), that when processed within the cell, combines with the RNA Induced Silencing Complex (RISC) resulting in specific mRNA degradation or translational blockage. To accomplish in vivo RNAi, day 9 hatched and fully expanded blastocysts are infected with the lentivirus for 4 to 5 h, and then surgically transferred to synchronized recipient uteri. Only the trophectoderm cells are infected by the replication deficient virus, leaving the inner cell mass unaltered, and we often obtain ~70% pregnancy rates following transfer of a single blastocyst. In vivo RNAi coupled with steady-state study of blood flow and nutrient uptake, transfer and utilization can now provide new insight into the physiological consequences of modifying the translation of specific genes expressed within the ruminant placenta.

Optimal placental function is required for offspring to reach their genetic potential in utero, and functional placental insufficiency not only results in increased offspring morbidity and mortality, but can impact production traits long-term. However, assessing placental function in vivo is technically demanding, and robust assessment of placental function requires cannulating both maternal and fetal vasculature in order to obtain arterial and venous blood samples simultaneously under non-stressed/non-anesthetized conditions. While feasible in cattle, this approach has been used more extensively in sheep, providing a thorough understanding of placental nutrient uptake, transport, and utilization in normal and compromised pregnancies. Previously, it has not been feasible to alter the abundance of specific gene products within the ruminant placenta, impairing the direct assessment of "cause and effect" relationships in vivo. However, recently methods have been developed to facilitate RNA interference (RNAi) within the placenta, effectively generating a deficiency in specific gene products, to examine the impact on pregnancy progression and outcome. While in vivo RNAi is feasible in a variety of species, in sheep it is being coupled with the aforementioned approaches assessing in vivo placental function, thereby providing new insight into the ramification of specific gene function within ruminant placenta.