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

Abstract

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.

Keywords: Fick principle; RNA interference; blood flow; nutrients; placenta; ruminant.

Figure 1.

Schematic representation of the placenta, uterine, and umbilical circulation, identifying catheter placement for sampling maternal and fetal blood under steady-state non-anesthetized/non-stressed conditions.

Figure 2.

Schematic representation of the uterine uptake, umbilical uptake, and placental metabolism of nutrients during late gestation in sheep, as assessed under steady-state non-anesthetized/non-stressed conditions.

Figure 3.

Schematic of the work flow for generating in vivo RNAi pregnancies, and their subsequent study.

Figure 4.

Schematic representation of the major physiological changes that occur in CSH RNAi pregnancies near-term that results in fetal growth restriction.

Figure 5.

Impact of fetal glucagon infusion on uterine artery concentrations of CSH/PL and estradiol. Samples were derived from the study reported by Cilvik et al. (2021), and demonstrate the timecourse by which fetal glucagon infusion impacts maternal CSH/PL (A) without impacting estradiol (B).

Figure 6.

Impact of in vivo CSH RNAi resulting in PI-FGR on umbilical glucose uptake in relationship to the maternofetal arterial glucose gradient. The intercept/elevation of the two regression lines are different (P ≤ 0.01) and the slope of the two lines tend to differ (P = 0.13).