Increasing maternal glucose concentrations is insufficient to restore placental glucose transfer in chorionic somatomammotropin RNA interference pregnancies.

We previously demonstrated impaired placental nutrient transfer in chorionic somatomammotropin (CSH) RNA interference (RNAi) pregnancies, with glucose transfer being the most impacted. Thus, we hypothesized that despite experimentally elevating maternal glucose, diminished umbilical glucose uptake would persist in CSH RNAi pregnancies, demonstrating the necessity of CSH for adequate placental glucose transfer. Trophectoderm of sheep blastocysts (9 days of gestational age; dGA) were infected with a lentivirus expressing either nontargeting control (CON RNAi; n = 5) or CSH-specific shRNA (CSH RNAi; n = 7) before transfer into recipient sheep. At 126 dGA, pregnancies were fitted with vascular catheters and underwent steady-state metabolic studies (3H2O transplacental diffusion) at 137 ± 0 dGA, before and during a maternal hyperglycemic clamp. Umbilical glucose and oxygen uptakes, as well as insulin and IGF1 concentrations, were impaired (P ≤ 0.01) in CSH RNAi fetuses and were not rescued by elevated maternal glucose. This is partially due to impaired uterine and umbilical blood flow (P ≤ 0.01). However, uteroplacental oxygen utilization was greater (P ≤ 0.05) during the maternal hyperglycemic clamp, consistent with greater placental oxidation of substrates. The relationship between umbilical glucose uptake and the maternal-fetal glucose gradient was analyzed, and while the slope (CON RNAi, Y = 29.54X +74.15; CSH RNAi, Y = 19.05X + 52.40) was not different, the y-intercepts and elevation were (P = 0.003), indicating reduced maximal glucose transport during maternal hyperglycemia. Together, these data suggested that CSH plays a key role in modulating placental metabolism that ultimately promotes maximal placental glucose transfer.NEW & NOTEWORTHY The current study demonstrated a novel, critical autocrine role for chorionic somatomammotropin in augmenting placental glucose transfer and maintaining placental oxidative metabolism. In pregnancies with CSH deficiency, excess glucose in maternal circulation is insufficient to overcome fetal hypoglycemia due to impaired placental glucose transfer and elevated placental metabolic demands. This suggests that perturbations in glucose transfer in CSH RNAi pregnancies are due to compromised metabolic efficiency along with reduced placental mass.

Fetal Hypoglycemia Induced by Placental SLC2A3-RNA Interference Alters Fetal Pancreas Development and Transcriptome at Mid-Gestation.

Glucose, the primary energy substrate for fetal oxidative processes and growth, is transferred from maternal to fetal circulation down a concentration gradient by placental facilitative glucose transporters. In sheep, SLC2A1 and SLC2A3 are the primary transporters available in the placental epithelium, with SLC2A3 located on the maternal-facing apical trophoblast membrane and SLC2A1 located on the fetal-facing basolateral trophoblast membrane. We have previously reported that impaired placental SLC2A3 glucose transport resulted in smaller, hypoglycemic fetuses with reduced umbilical artery insulin and glucagon concentrations, in addition to diminished pancreas weights. These findings led us to subject RNA derived from SLC2A3-RNAi (RNA interference) and NTS-RNAi (non-targeting sequence) fetal pancreases to qPCR followed by transcriptomic analysis. We identified a total of 771 differentially expressed genes (DEGs). Upregulated pathways were associated with fat digestion and absorption, particularly fatty acid transport, lipid metabolism, and cholesterol biosynthesis, suggesting a potential switch in energetic substrates due to hypoglycemia. Pathways related to molecular transport and cell signaling in addition to pathways influencing growth and metabolism of the developing pancreas were also impacted. A few genes directly related to gluconeogenesis were also differentially expressed. Our results suggest that fetal hypoglycemia during the first half of gestation impacts fetal pancreas development and function that is not limited to ß cell activity.

Uptake of Phosphate, Calcium, and Vitamin D by the Pregnant Uterus of Sheep in Late Gestation: Regulation by Chorionic Somatomammotropin Hormone.

Minerals are required for the establishment and maintenance of pregnancy and regulation of fetal growth in mammals. Lentiviral-mediated RNA interference (RNAi) of chorionic somatomammotropin hormone (CSH) results in both an intrauterine growth restriction (IUGR) and a non-IUGR phenotype in sheep. This study determined the effects of CSH RNAi on the concentration and uptake of calcium, phosphate, and vitamin D, and the expression of candidate mRNAs known to mediate mineral signaling in caruncles (maternal component of placentome) and cotyledons (fetal component of placentome) on gestational day 132. CSH RNAi Non-IUGR pregnancies had a lower umbilical vein−umbilical artery calcium gradient (p < 0.05) and less cotyledonary calcium (p < 0.05) and phosphate (p < 0.05) compared to Control RNAi pregnancies. CSH RNAi IUGR pregnancies had less umbilical calcium uptake (p < 0.05), lower uterine arterial and venous concentrations of 25(OH)D (p < 0.05), and trends for lower umbilical 25(OH)D uptake (p = 0.059) compared to Control RNAi pregnancies. Furthermore, CSH RNAi IUGR pregnancies had decreased umbilical uptake of calcium (p < 0.05), less uterine venous 25(OH)D (vitamin D metabolite; p = 0.055), lower caruncular expression of SLC20A2 (sodium-dependent phosphate transporter; p < 0.05) mRNA, and lower cotyledonary expression of KL (klotho; p < 0.01), FGFR1 (fibroblast growth factor receptor 1; p < 0.05), FGFR2 (p < 0.05), and TRPV6 (transient receptor potential vanilloid member 6; p < 0.05) mRNAs compared to CSH RNAi Non-IUGR pregnancies. This study has provided novel insights into the regulatory role of CSH for calcium, phosphate, and vitamin D utilization in late gestation.

Impact of Placental SLC2A3 Deficiency during the First-Half of Gestation.

In the ruminant placenta, glucose uptake and transfer are mediated by facilitative glucose transporters SLC2A1 (GLUT1) and SLC2A3 (GLUT3). SLC2A1 is located on the basolateral trophoblast membrane, whereas SLC2A3 is located solely on the maternal-facing, apical trophoblast membrane. While SLC2A3 is less abundant than SLC2A1, SLC2A3 has a five-fold greater affinity and transport capacity. Based on its location, SLC2A3 likely plays a significant role in the uptake of glucose into the trophoblast. Fetal hypoglycemia is a hallmark of fetal growth restriction (FGR), and as such, any deficiency in SLC2A3 could impact trophoblast glucose uptake and transfer to the fetus, thus potentially setting the stage for FGR. By utilizing in vivo placenta-specific lentiviral-mediated RNA interference (RNAi) in sheep, we were able to significantly diminish (p ≤ 0.05) placental SLC2A3 concentration, and determine the impact at mid-gestation (75 dGA). In response to SLC2A3 RNAi (n = 6), the fetuses were hypoglycemic (p ≤ 0.05), exhibited reduced fetal growth, including reduced fetal pancreas weight (p ≤ 0.05), which was associated with reduced umbilical artery insulin and glucagon concentrations, when compared to the non-targeting sequence (NTS) RNAi controls (n = 6). By contrast, fetal liver weights were not impacted, nor were umbilical artery concentrations of IGF1, possibly resulting from a 70% increase (p ≤ 0.05) in umbilical vein chorionic somatomammotropin (CSH) concentrations. Thus, during the first half of gestation, a deficiency in SLC2A3 results in fetal hypoglycemia, reduced fetal development, and altered metabolic hormone concentrations. These results suggest that SLC2A3 may be the rate-limiting placental glucose transporter during the first-half of gestation in sheep.

Impact of chorionic somatomammotropin RNA interference on uterine blood flow and placental glucose uptake in the absence of intrauterine growth restriction.

Chorionic somatomammotropin (CSH) is one of the most abundantly produced placental hormones, yet its exact function remains elusive. Near-term [135 days of gestational age (dGA)], CSH RNA interference (RNAi) results in two distinct phenotypes: 1) pregnancies with intrauterine growth restriction (IUGR), and 2) pregnancies with normal fetal and placental weights. Here, we report the physiological changes in CSH RNAi pregnancies without IUGR. The trophectoderm of hatched blastocysts (9 dGA) were infected with lentiviral-constructs expressing either a scrambled control (Control RNAi) or CSH-specific shRNA (CSH RNAi), prior to transfer into synchronized recipient ewes. At 126 dGA, Control RNAi (n = 6) and CSH RNAi (n = 6) pregnancies were fitted with maternal and fetal catheters. Uterine and umbilical blood flows were measured at 132 dGA and nutrient uptakes were calculated by the Fick's principle. Control RNAi and CSH RNAi pregnancies were compared by analysis of variance, and significance was set at P ≤ 0.05. Absolute (mL/min) and relative (mL/min/kg fetus) uterine blood flows were reduced (P ≤ 0.05) in CSH RNAi pregnancies, but umbilical flows were not impacted. The uterine artery-to-vein glucose gradient (mmol/L) was significantly (P ≤ 0.05) increased. The uteroplacental glucose uptake (µmoL/min/kg placenta) was increased (P ≤ 0.05), whereas umbilical glucose uptake (µmoL/min/kg fetus) was reduced. Our results demonstrate that CSH RNAi has significant physiological ramifications, even in the absence of IUGR, and comparing CSH RNAi pregnancies exhibiting both IUGR and non-IUGR phenotypes may help determine the direct effects of CSH and its potential impact on fetal development.

MicroRNA-Mediated Gene Regulatory Mechanisms in Mammalian Female Reproductive Health.

Mammalian reproductive health affects the entire reproductive cycle starting with the ovarian function through implantation and fetal growth. Various environmental and physiological factors contribute to disturbed reproductive health status leading to infertility problems in mammalian species. In the last couple of decades a significant number of studies have been conducted to investigate the transcriptome of reproductive tissues and organs in relation to the various reproductive health issues including endometritis, polycystic ovarian syndrome (PCOS), intrauterine growth restriction (IUGR), preeclampsia, and various age-associated reproductive disorders. Among others, the post-transcriptional regulation of genes by small noncoding miRNAs contributes to the observed transcriptome dysregulation associated with reproductive pathophysiological conditions. MicroRNAs as a class of non-coding RNAs are also known to be involved in various pathophysiological conditions either in cellular cytoplasm or they can be released to the extracellular fluid via membrane-bounded extracellular vesicles and proteins. The present review summarizes the cellular and extracellular miRNAs and their association with the etiology of major reproductive pathologies including PCOS, endometritis, IUGR and age-associated disorders in various mammalian species.

CSH RNA Interference Reduces Global Nutrient Uptake and Umbilical Blood Flow Resulting in Intrauterine Growth Restriction.

Deficiency of the placental hormone chorionic somatomammotropin (CSH) can lead to the development of intrauterine growth restriction (IUGR). To gain insight into the physiological consequences of CSH RNA interference (RNAi), the trophectoderm of hatched blastocysts (nine days of gestational age; dGA) was infected with a lentivirus expressing either a scrambled control or CSH-specific shRNA, prior to transfer into synchronized recipient sheep. At 90 dGA, umbilical hemodynamics and fetal measurements were assessed by Doppler ultrasonography. At 120 dGA, pregnancies were fitted with vascular catheters to undergo steady-state metabolic studies with the 3H2O transplacental diffusion technique at 130 dGA. Nutrient uptake rates were determined and tissues were subsequently harvested at necropsy. CSH RNAi reduced (p ≤ 0.05) both fetal and uterine weights as well as umbilical blood flow (mL/min). This ultimately resulted in reduced (p ≤ 0.01) umbilical IGF1 concentrations, as well as reduced umbilical nutrient uptakes (p ≤ 0.05) in CSH RNAi pregnancies. CSH RNAi also reduced (p ≤ 0.05) uterine nutrient uptakes as well as uteroplacental glucose utilization. These data suggest that CSH is necessary to facilitate adequate blood flow for the uptake of oxygen, oxidative substrates, and hormones essential to support fetal and uterine growth.

LIN28-let-7 axis regulates genes in immortalized human trophoblast cells by targeting the ARID3B-complex.

Abnormal placental development is one of the main etiological factors for intrauterine growth restriction (IUGR). Here, we show that LIN28A and LIN28B are significantly lower and lethal-7 (let-7) microRNAs (miRNAs) significantly higher in term human IUGR vs. normal placentas. We hypothesize that let-7 miRNAs regulate genes with known importance for human placental development [high-mobility group AT-hook 1 (HMGA1), transcriptional regulator Myc-like (c-myc), vascular endothelial growth factor A (VEGF-A), and Wnt family member 1 (WNT1)] by targeting the AT-rich interacting domain (ARID)-3B complex. ACH-3P cells with LIN28A and LIN28B knockout (DKOs) significantly increased let-7 miRNAs, leading to significantly decreased ARID3A, ARID3B, and lysine demethylase 4C (KDM4C). Similarly, Sw.71 cells overexpressing LIN28A and LIN28B (DKIs) significantly decreased let-7 miRNAs, leading to significantly increased ARID3A, ARID3B, and KDM4C. In ACH-3P cells, ARID3A, ARID3B, and KDM4C make a triprotein complex [triprotein complex comprising ARID3A, ARID3B, and KDM4C (ARID3B-complex)] that binds the promoter regions of HMGA1, c-MYC, VEGF-A, and WNT1. ARID3B knockout in ACH-3P cells disrupted the ARID3B-complex, leading to a significant decrease in HMGA1, c-MYC, VEGF-A, and WNT1. DKOs had a significant reduction, whereas DKIs had a significant increase in HMGA1, c-MYC, VEGF-A, and WNT1, potentially due to regulation by the ARID3B-complex. This is the first study showing regulation of let-7 targets in immortalized human trophoblast cells by the ARID3B-complex.-Ali, A., Anthony, R. V., Bouma, G. J., Winger, Q. A. LIN28-let-7 axis regulates genes in immortalized human trophoblast cells by targeting the ARID3B-complex.

The Role of LIN28-let-7-ARID3B Pathway in Placental Development.

Placental disorders are a major cause of pregnancy loss in humans, and 40-60% of embryos are lost between fertilization and birth. Successful embryo implantation and placental development requires rapid proliferation, invasion, and migration of trophoblast cells. In recent years, microRNAs (miRNAs) have emerged as key regulators of molecular pathways involved in trophoblast function. A miRNA binds its target mRNA in the 3'-untranslated region (3'-UTR), causing its degradation or translational repression. Lethal-7 (let-7) miRNAs induce cell differentiation and reduce cell proliferation by targeting proliferation-associated genes. The oncoprotein LIN28 represses the biogenesis of mature let-7 miRNAs. Proliferating cells have high LIN28 and low let-7 miRNAs, whereas differentiating cells have low LIN28 and high let-7 miRNAs. In placenta, low LIN28 and high let-7 miRNAs can lead to reduced proliferation of trophoblast cells, resulting in abnormal placental development. In trophoblast cells, let-7 miRNAs reduce the expression of proliferation factors either directly by binding their mRNA in 3'-UTR or indirectly by targeting the AT-rich interaction domain (ARID)3B complex, a transcription-activating complex comprised of ARID3A, ARID3B, and histone demethylase 4C (KDM4C). In this review, we discuss regulation of trophoblast function by miRNAs, focusing on the role of LIN28-let-7-ARID3B pathway in placental development.

Trophectoderm-Specific Knockdown of LIN28 Decreases Expression of Genes Necessary for Cell Proliferation and Reduces Elongation of Sheep Conceptus.

LIN28 inhibits let-7 miRNA maturation which prevents cell differentiation and promotes proliferation. We hypothesized that the LIN28-let-7 axis regulates proliferation-associated genes in sheep trophectoderm in vivo. Day 9-hatched sheep blastocysts were incubated with lentiviral particles to deliver shRNA targeting LIN28 specifically to trophectoderm cells. At day 16, conceptus elongation was significantly reduced in LIN28A and LIN28B knockdowns. Let-7 miRNAs were significantly increased and IGF2BP1-3, HMGA1, ARID3B, and c-MYC were decreased in trophectoderm from knockdown conceptuses. Ovine trophoblast (OTR) cells derived from day 16 trophectoderm are a useful tool for in vitro experiments. Surprisingly, LIN28 was significantly reduced and let-7 miRNAs increased after only a few passages of OTR cells, suggesting these passaged cells represent a more differentiated phenotype. To create an OTR cell line more similar to day 16 trophectoderm we overexpressed LIN28A and LIN28B, which significantly decreased let-7 miRNAs and increased IGF2BP1-3, HMGA1, ARID3B, and c-MYC compared to control. This is the first study showing the role of the LIN28-let-7 axis in trophoblast proliferation and conceptus elongation in vivo. These results suggest that reduced LIN28 during early placental development can lead to reduced trophoblast proliferation and sheep conceptus elongation at a critical period for successful establishment of pregnancy.

Development of ovine chorionic somatomammotropin hormone-deficient pregnancies.

Intrauterine growth restriction (IUGR) is a leading cause of neonatal mortality and morbidity. Chorionic somatomammotropin hormone (CSH), a placenta-specific secretory product found at high concentrations in maternal and fetal circulation throughout gestation, is significantly reduced in human and sheep IUGR pregnancies. The objective of this study was to knock down ovine CSH (oCSH) expression in vivo using lentiviral-mediated short-hairpin RNA to test the hypothesis that oCSH deficiency would result in IUGR of near-term fetal lambs. Three different lentiviral oCSH-targeting constructs were used and compared with pregnancies (n = 8) generated with a scrambled control (SC) lentiviral construct. Pregnancies were harvested at 135 days of gestation. The most effective targeting sequence, "target 6" (tg6; n = 8), yielded pregnancies with significant reductions (P ≤ 0.05) in oCSH mRNA (50%) and protein (38%) concentrations, as well as significant reductions (P ≤ 0.05) in placental (52%) and fetal (32%) weights compared with the SC pregnancies. Fetal liver weights were reduced 41% (P ≤ 0.05), yet fetal liver insulin-like growth factor-I (oIGF1) and -II mRNA concentrations were reduced (P ≤ 0.05) 82 and 71%, respectively, and umbilical artery oIGF1 concentrations were reduced 62% (P ≤ 0.05) in tg6 pregnancies. Additionally, fetal liver oIGF-binding protein (oIGFBP) 2 and oIGFBP3 mRNA concentrations were reduced (P ≤ 0.05), whereas fetal liver oIGFBP1 mRNA concentration was not impacted nor was maternal liver oIGF and oIGFBP mRNA concentrations or uterine artery oIGF1 concentrations (P ≥ 0.10). Based on our results, it appears that oCSH deficiency does result in IUGR, by impacting placental development as well as fetal liver development and function.

Embryo mortality in Isg15-/- mice is exacerbated by environmental stress.

The interferon-stimulated gene 15 (Isg15) encodes a ubiquitin-like protein that is induced in the endometrium by pregnancy in mice, humans, and ruminants. Because ISG15 is a component of the innate immune system, we hypothesized that development of the embryo, fetus, and postnatal pup may be impaired in mice lacking Isg15 (Isg15(-/-)) and that this development would be further impaired in response to environmental insults such as hypoxia. The number of implantation sites, resorption sites, dead embryos, and the changes in overall gross morphology of the uterus were evaluated in Isg15(-/-) mice on Days 7.5 and 12.5 postcoitum (dpc). Postnatal development also was monitored from birth to 12 wk of age. On 7.5 dpc, the number of implantation sites and serum progesterone concentrations were similar. However, embryo mortality increased (P < 0.05) in Isg15(-/-) dams by 12.5 dpc, resulting in smaller litter sizes (4.26 ± 0.21 embryos; n = 83 litters) compared to Isg15(+/+) females (7.78 ± 0.29 pups; n = 47 litters). Embryo mortality in Isg15(-/-) mice was further exacerbated to 70% when dams were stressed through housing under hypoxic conditions (PB = 445 mmHg; 6.5-12.5 dpc). Transmission electron microscopy revealed lesions in antimesometrial decidua as well as trophoblast cells adjacent to decidual cells on 7.5 dpc. ISG15 was localized to mesometrial decidua on 7.5 dpc. By 12.5 dpc, ISG15 was intensely localized to the labyrinth of the placenta. By 7.5 dpc, uterine natural killer cell migration into the mesometrial pole was diminished by 65% and was less prevalent in Isg15(-/-) compared to Isg15(+/+) deciduum. Postnatal growth rate of offspring that survived to birth from Isg15(-/-) and Isg15(+/+) dams was not different. Embryo mortality occurs in pregnant Isg15(-/-) mice, is exacerbated by environmental insults like maternal hypoxia, and might result from impaired early decidualization, vascular development, and formation of the labyrinth.

Temporal Release, Paracrine and Endocrine Actions of Ovine Conceptus-Derived Interferon-Tau During Early Pregnancy.

The antiviral activity of interferon (IFN) increases in uterine vein serum (UVS) during early pregnancy in sheep. This antiviral activity in UVS collected on Day 15 of pregnancy is blocked by anti-IFN-tau (anti-IFNT) antibodies. Conceptus-derived IFNT was hypothesized to induce IFN-stimulated gene (ISG) expression in endometrium and extrauterine tissues during pregnancy. To test this hypothesis, blood was collected from ewes on Days 12-16 of the estrous cycle or pregnancy. Serum progesterone was >1.7 ng/ml in pregnant (P) and nonpregnant (NP) ewes until Day 13, then declined to <0.6 ng/ml by Day 15 in NP ewes. A validated IFNT radioimmunoassay detected IFNT in uterine flushings (UFs) on Days 13-16 and in UVS on Days 15-16 of pregnancy. IFNT detection in UF correlated with paracrine induction of ISGs in the endometrium and occurred prior to the inhibition of estrogen receptor 1 and oxytocin receptor expression in uterine epithelia on Day 14 of pregnancy. Induction of ISG mRNAs in corpus luteum (CL) and liver tissue occurred by Day 14 and in peripheral blood mononuclear cells by Day 15 in P ewes. Expression of mRNAs for IFN signal transducers and ISGs were greater in the CL of P than that of NP ewes on Day 14. It is concluded that: 1) paracrine actions of IFNT coincide with detection of IFNT in UF; 2) endocrine action of IFNT ensues through induction of ISGs in peripheral tissues; and 3) IFNT can be detected in UVS, but not until Days 15-16 of pregnancy, which may be limited by the sensitivity of the IFNT radioimmunoassay.

Elevated maternal cortisol leads to relative maternal hyperglycemia and increased stillbirth in ovine pregnancy.

In normal pregnancy, cortisol increases; however, further pathological increases in cortisol are associated with maternal and fetal morbidities. These experiments were designed to test the hypothesis that increased maternal cortisol would increase maternal glucose concentrations, suppress fetal growth, and impair neonatal glucose homeostasis. Ewes were infused with cortisol (1 mg·kg(-1)·day(-1)) from day 115 of gestation to term; maternal glucose, insulin, ovine placental lactogen, estrone, progesterone, nonesterified free fatty acids (NEFA), ß-hydroxybutyrate (BHB), and electrolytes were measured. Infusion of cortisol increased maternal glucose concentration and slowed the glucose disappearance after injection of glucose; maternal infusion of cortisol also increased the incidence of fetal death at or near parturition. The design of the study was altered to terminate the study prior to delivery, and post hoc analysis of the data was performed to test the hypothesis that maternal metabolic factors predict the fetal outcome. In cortisol-infused ewes that had stillborn lambs, plasma insulin was increased relative to control ewes or cortisol-infused ewes with live lambs. Maternal cortisol infusion did not alter maternal food intake or plasma NEFA, BHB, estrone, progesterone or placental lactogen concentrations, and it did not alter fetal body weight, ponderal index, or fetal organ weights. Our study suggests that the adverse effect of elevated maternal cortisol on pregnancy outcome may be related to the effects of cortisol on maternal glucose homeostasis, and that chronic maternal stress or adrenal hypersecretion of cortisol may create fetal pathophysiology paralleling some aspects of maternal gestational diabetes.

The Impact of SLC2A8 RNA Interference on Glucose Uptake and the Transcriptome of Human Trophoblast Cells.

While glucose is the primary fuel for fetal growth, the placenta utilizes the majority of glucose taken up from the maternal circulation. Of the facilitative glucose transporters in the placenta, SLC2A8 (GLUT8) is thought to primarily function as an intracellular glucose transporter; however, its function in trophoblast cells has not been determined. To gain insight into the function of SLC2A8 in the placenta, lentiviral-mediated RNA interference (RNAi) was performed in the human first-trimester trophoblast cell line ACH-3P. Non-targeting sequence controls (NTS RNAi; n = 4) and SLC2A8 RNAi (n = 4) infected ACH-3P cells were compared. A 79% reduction in SLC2A8 mRNA concentration was associated with an 11% reduction (p ≤ 0.05) in ACH-3P glucose uptake. NTS RNAi and SLC2A8 RNAi ACH-3P mRNA were subjected to RNAseq, identifying 1525 transcripts that were differentially expressed (|log2FC| > 1 and adjusted p-value < 0.05), with 273 transcripts derived from protein-coding genes, and the change in 10 of these mRNAs was validated by real-time qPCR. Additionally, there were 147 differentially expressed long non-coding RNAs. Functional analyses revealed differentially expressed genes involved in various metabolic pathways associated with cellular respiration, oxidative phosphorylation, and ATP synthesis. Collectively, these data indicate that SLC2A8 deficiency may impact placental uptake of glucose, but that its likely primary function in trophoblast cells is to support cellular respiration. Since the placenta oxidizes the majority of the glucose it takes up to support its own metabolic needs, impairment of SLC2A8 function could set the stage for functional placental insufficiency.

Role of LIN28A in mouse and human trophoblast cell differentiation.

Proper regulation of trophoblast proliferation, differentiation, and function are critical for placenta development and function. The RNA-binding protein, LIN28A, has been well characterized as a potent regulator of differentiation in embryonic stem cells; however, little is known about the function of LIN28A in the placenta. We assessed LIN28A in vitro using mouse trophoblast stem (mTS) cells and human trophoblast cells (ACH-3P). We observed that LIN28A decreased and let-7 miRNA increased when mTS cells were induced to differentiate into mouse trophoblast giant cells (mTGCs) upon the removal of FGF4, heparin and conditioned medium. Similarly, we observed that LIN28A decreased in ACH-3P cells induced to syncytialize with forskolin treatment. To assess LIN28A in vivo we examined Embryonic Day 11.5 mouse placenta and observed abundant LIN28A in the chorioallantoic interface and labyrinth layer, with little LIN28A staining in spongiotrophoblast or differentiated mTGCs. Additionally, shRNA-mediated LIN28A knockdown in ACH-3P cells resulted in increased spontaneous syncytialization, and increased levels of syncytiotrophoblast markers hCG, LGALS13, and ERVW-1 mRNA. Additionally, targeted degradation of LIN28A mRNA increased responsiveness to forskolin-induced differentiation. In contrast, targeted degradation of Lin28a mRNA in mTS cells did not alter cell phenotype when maintained under proliferative culture conditions. Together, these data establish that LIN28A has a functional role in regulating trophoblast differentiation and function, and that loss of LIN28A in human trophoblast is sufficient to induce differentiation, but does not induce differentiation in the mouse.

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.

Trophectoderm Transcriptome Analysis in LIN28 Knockdown Ovine Conceptuses Suggests Diverse Roles of the LIN28-let-7 Axis in Placental and Fetal Development.

The proper conceptus elongation in ruminants is critical for the successful placentation and establishment of pregnancy. We have previously shown that the trophectoderm-specific knockdown of LIN28A/B in day 9 ovine blastocysts resulted in increased let-7 miRNAs and reduced conceptus elongation at day 16 of gestation. In this current study, by transcriptome analysis of LIN28A knockdown (AKD) or LIN28B knockdown (BKD) trophectoderm (TE), we explored the downstream target genes of the LIN28-let-7 axis and their roles in the placental and fetal development. We identified 449 differentially expressed genes (DEGs) in AKD TE and 1214 DEGs in BKD TE compared to non-targeting control (NTC). Our analysis further revealed that 210 downregulated genes in AKD TE and 562 downregulated genes in BKD TE were the potential targets of let-7 miRNAs. Moreover, 16 downregulated genes in AKD TE and 57 downregulated and 7 upregulated genes in BKD TE were transcription factors. The DEGs in AKD and BKD TE showed enrichment in the biological processes and pathways critical for placental development and function, and fetal development and growth. The results of this study suggest the potential roles of the LIN28-let-7 axis in placental and fetal development beyond its involvement in trophoblast proliferation and conceptus elongation.

The chemokine receptor CXCR4 and its ligand CXCL12 are activated during implantation and placentation in sheep.

BACKGROUND: The progression of implantation and placentation in ruminants is complex and is regulated by interplay between sex steroids and local signaling molecules, many of which have immune function. Chemokines and their receptors are pivotal factors in implantation and vascularization of the placenta. Based on known critical roles for chemokine receptor 4 (CXCR4) during early pregnancy in other species, we hypothesized that CXCR4 and its ligand CXCL12 would increase in the endometrium and conceptus in response to implantation in ewes. The objectives of the current study were to determine if CXCL12 and CXCR4 were upregulated in: endometrium from pregnant compared to non-pregnant ewes and in, conceptuses, cotyledons, caruncles and intercaruncular tissue. METHODS: Tissues were collected from sheep on Days 12, 13, 14, and 15 of either the estrous cycle or pregnancy and from pregnant ewes on Days 35 and 50. Blood samples from jugular and uterine vein were also collected on all days. Conceptuses were collected from mature ewes on Days 13, 15, 16, 17, 21 and 30 of gestation. Real time PCR was used to determine relative mRNA concentrations for CXCL12 and CXCR4 and Western blot analysis was employed to confirm protein concentration. RESULTS: Differences described are P < 0.05. In the endometrium, CXCR4 mRNA and protein was greater on Day 15 of pregnancy compared to the estrous cycle. CXCL12 and CXCR4 mRNA in conceptuses was greater on Days 21 and 30 compared to earlier days. CXCL12 mRNA was greater in cotyledons on Day 35 compared to Day 50. On Day 35 of gestation, CXCR4 was greater compared to Day 50 in caruncle and intercaruncular tissue. White blood cells obtained from jugular and uterine vein collection had the greatest mRNA concentration of CXCL12 on Day 35 of pregnancy. CONCLUSIONS: A comprehensive analysis of CXCL12 and CXCR4 expression in fetal and maternal tissues during early pregnancy is reported with noteworthy differences occurring during implantation and placentation in sheep. We interpreted these data to mean that the CXCL12/CXCR4 pathway is activated during implantation and placentation in sheep and is likely playing a role in the communication between trophoblast cells and the maternal endometrium.