Role of the placenta in developmental programming: Observations from models using large animals.

Developmental programming, which proposes that "insults" or "stressors" during intrauterine or postnatal development can have not only immediate but also long-term consequences for healthy and productivity, has emerged as a major biological principle, and based on studies in many animal species also seems to be a universal phenomenon. In eutherians, the placenta appears to be programmed during its development, which has consequences for fetal growth and development throughout pregnancy, and likewise has long-term consequences for postnatal development, leading to programming of organ function of the offspring even into adulthood. This review summarizes our current understanding of the placenta's role in developmental programming, the mechanisms involved, and the challenges remaining.

The effects of maternal nutrition on the messenger ribonucleic acid expression of neutral and acidic amino acid transporters in bovine uteroplacental tissues from day sixteen to fifty of gestation.

We hypothesized that both day of gestation and maternal nutrition would alter the relative mRNA expression of neutral and acid AA transporters , , , , and . Crossbred Angus heifers ( = 49) were synchronized, bred via AI, assigned to nutritional treatment (100% of NRC requirements for 0.45 kg/d gain [control heifers {CON}] and 60% of CON [restricted heifers {RES}]), and ovariohysterectomized on d 16, 34, or 50 of gestation ( = 6 to 9/d). Nonbred, nonpregnant (NB-NP) controls were ovariohysterectomized on d 16 of the estrous cycle ( = 6) after synchronization. The resulting arrangement was a 2 × 3 factorial + 1 (CON vs. RES × d 16, 34, or 50 + NB-NP controls). Tissues collected included caruncular endometrium (CAR), intercaruncular endometrium (ICAR), fetal membranes (FM; chorioallantois; d 16 and 34), cotyledonary placenta (COT; d 50 only), intercotyledonary placenta (ICOT; d 50 only), and amnion (AMN; d 50 only]). Relative expression of , , , , and was determined for each tissue using NB-NP CAR and NB-NP ICAR tissues for the baseline; for FM, endometrium from NB-NP controls served as the baseline. In CAR, no day × treatment interaction was observed ( > 0.05). However, day of gestation affected relative expression of , where expression on d 16 was greater ( < 0.01) than expression on d 34 and 50. Additionally, relative expression of and was greater ( ≤ 0.05) in pregnant heifers compared with NB-NP heifers. For ICAR, was influenced by a day × treatment interaction ( < 0.01), where expression in d 16 RES was greater ( ≤ 0.05) than that of any other day or nutritional treatment. Furthermore, expression in d 16 CON was greater ( ≤ 0.05) than that in d 50 RES, with those in d 34 CON and RES and d 50 CON being intermediate. In addition, was affected by day of gestation, where expression on d 16 was greater ( < 0.01) than that on d 34 and 50. A day × treatment interaction was not observed ( > 0.05) in FM; however, expression on d 34 was greater ( = 0.02) than on d 50, with that on d 16 being intermediate. Day of gestation also affected expression of , where expression on d 34 and 50 was greater ( < 0.01) than that on d 16. These data support our hypothesis in that both day of gestation and maternal nutrition affected the relative mRNA expression of AA transporter in ICAR, whereas day of gestation has a greater effect on the relative mRNA expression of other neutral and acidic AA transporters in the various tissues studied.

Impacts of maternal nutrition on uterine and placental vascularity and mRNA expression of angiogenic factors during the establishment of pregnancy in beef heifers.

We hypothesized that maternal nutrient restriction starting at the time of breeding would influence placental vascular development and gene expression of angiogenic factors during the first 50 d of gestation in beef heifers. Commercial Angus crossbred heifers (n = 49) were maintained on a total mixed ration and supplemented with dried distillers grains with solubles. All heifers were subject to 5-d CO-Synch + CIDR estrous synchronization protocol, AI to a single Angus sire, and randomly assigned to dietary treatments. One half were assigned to control diet (CON) targeted to gain 0.45 kg/d and the remaining half were assigned to restricted diet (RES), which received 60% of CON. Heifers were subjected to ovariohysterectomy on d 16, 34, or 50 of gestation. Utero-placental tissues were obtained from the uterine horns ipsilateral and contralateral to the corpus luteum and separated into maternal caruncle (CAR); maternal endometrium, inter-caruncle (ICAR), and fetal membranes (FM). After collection, all tissues were snap frozen and stored at -80°C. There were no treatment × stage of gestation interactions (P >0.13) on the mRNA expression of vascular endothelial growth factor (VEGF) or endothelial nitric oxide synthase (eNOS). Heifers on CON treatment had greater (P = 0.03) expression of VEGF compared with RES heifers in NP-ICAR. On d 50 expression of eNOS was increased (P = 0.05) compared with d 16 in P-CAR. Expression of eNOS mRNA was decreased (P = 0.04) on d 16 compared with d 34 and 50 in CON heifer. Gene expression of eNOS was increased (P < 0.001) in the pregnant uterine horn compared with the NP uterine horn on d 34 and 50. Expression of eNOS was also increased (P < 0.003) on d 34 and 50 in the pregnant uterine horn compared with FM. There was a maternal nutritional plane × stage of gestation interaction (P = 0.01) on the vascular ratio (vascular volume/tissue volume) in maternal tissues. The RES heifers had a greater vascular ratio on d 16 compared with d 34 and 50; whereas, CON heifers had a greater vascular ratio on d 34 compared with d 16 and 50. In the NP uterine horn, there was also an increase (P = 0.02) in vascular volume of FM from CON heifers compared with FM from RES heifers. We conclude that maternal nutrient restriction did alter both vascularity and mRNA expression of angiogenic factor in utero-placental tissues during the establishment of pregnancy in first parity beef heifers.

Endogenous retroviral gene elements (syncytin-Rum1 and BERV-K1), interferon-τ, and pregnancy associated glycoprotein-1 are differentially expressed in maternal and fetal tissues during the first 50 days of gestation in beef heifers.

We hypothesized that the endogenous retroviruses [ERV: syncytin-Rum1 and (BERV-K1)], and pregnancy hormones [interferon-τ (IFN-τ), and pregnancy associated glycoprotein-1 (PAG-1)] would be differentially expressed whereas progesterone and insulin concentrations in maternal blood would remain steady during early gestation. To test this hypothesis Angus crossbred heifers (n = 46; ∼15 mo of age; BW = 363 ± 35 kg) were fed native grass hay, supplemented with cracked corn to gain 0.3 kg/d, and given ad libitum access to water. All heifers were subjected to a 5-d CO-Synch + CIDR estrous synchronization protocol and AI (breeding = d 0). Ovariohysterectomies were performed on d 16, 22, 28, 34, 40, and 50 of gestation and at d 16 of the estrous cycle for non-pregnant (NP) controls. Utero-placental tissues [maternal caruncle (CAR); maternal intercaruncular endometrium (ICAR); and fetal membranes, (FM, chorion on d 16, chorioallantois on d 22 to 50)] were collected from the uterine horn ipsilateral to the corpus luteum (CL). Tissues were flash frozen and stored at -80°C. Expression of mRNA was evaluated using qPCR. In CAR, syncytin-Rum1 expression was greater (P < 0.01) on d 50 (81.5-fold) compared with NP controls or any other day of early pregnancy. In contrast, syncytin-Rum1 expression in I-CAR only tended (P = 0.09) to change across days of early pregnancy and did not differ (P = 0.27) in FM tissues. In CAR, the expression of BERV-K1 was not different (P > 0.79) at d 16 and 22, was intermediate at d 28, 34, and 40, and was greatest on d 50 (108-fold increase compared with NP). Expression of BERV-K1 in FM was increased (P < 0.01) on d 28, 34, and 50 compared with NP controls, but at d 40 did not differ from NP controls. The mRNA expression of IFN-τ in FM at d 22 was greater (P < 0.01) than all other days of gestation. In CAR, expression of PAG-1 increased (P < 0.001) dramatically on d 40 (20,000-fold) and d 50 (86,000-fold) compared with NP heifers (P < 0.01). In ICAR, expression of PAG-1 was greater (P < 0.05) on d 28 and 40 (fold increases of 113 and 102, respectively, compared with NP). Insulin concentrations were not different (P = 0.53) but progesterone was greater (P < 0.01) on d 16, 22, 28, 34, and 40 compared with d 50 of gestation. These data confirm differential ERV, IFN-τ, and PAG-1 gene expression during critical time points of early gestation in utero-placental tissues.

Maternal nutrition and stage of early pregnancy in beef heifers: Impacts on expression of glucose, fructose, and cationic amino acid transporters in utero-placental tissues.

We hypothesized that maternal nutrition and day of gestation would impact utero-placental mRNA expression of the nutrient transporters , , , , and in beef heifers. Crossbred Angus heifers (n = 49) were estrous synchronized, bred via AI, assigned to nutritional treatment (CON = 100% of NRC requirements for 0.45 kg/d gain and RES = 60% of CON) and ovariohysterectomized on d 16, 34, or 50 of gestation (n = 6 to 9/d); Non-bred, non-pregnant (NB-NP) controls were fed the CON diet, not bred, and were ovariohysterectomized on d 16 of the synchronized estrous cycle = 6). The resulting arrangement of treatments was a 2 × 3 factorial + 1 (CON vs. RES × d 16, 34, or 50 + NB-NP controls). Caruncle (CAR), intercaruncular endometrium (ICAR), and fetal membranes (FM [chorioallantois]), were obtained from the pregnant uterine horn (the uterine horn containing the conceptus) immediately after ovariohysterectomy. On d 50 cotyledons (COT), intercotyledonary placenta (ICOT) and amnion (AMN) were also collected. Relative expression of nutrient transporters was determined for each tissue utilizing NB-NP-CAR and NB-NP-ICAR tissues as the baseline. For FM, NB-NP endometrium served as the baseline. There was no interaction of day × treatment ( ≥ 0.20) for any genes in CAR. However, CAR expression of was greater ( < 0.01) on d 16 compared with d 34 and 50, and , , and were greater ( ≤ 0.05) on d 34 compared with d 16 and 50. In ICAR, was the only gene to be influenced by the day × treatment interaction ( = 0.01), being greater in d 50 CON compared with d 34 CON and d 16 and 50 RES. In ICAR, expression of was greater ( < 0.01) on d 16 compared with d 34, and expression of was greater ( < 0.01) on d 34 and 50 compared with d 16. In FM, expression of was greater ( = 0.04) on d 16 compared with d 50 of gestation, and expression of was greater ( < 0.01) on d 34 and 50 compared with d 16. On d 50, expression of , , and expression were all greater ( < 0.05) in AMN compared with COT and ICOT, and expression of was greater ( < 0.01) in ICOT compared with COT and AMN. These data indicate that day was a more influential factor for mRNA expression of utero-placental glucose and cationic AA transporters than maternal nutritional status in heifers during early pregnancy.

RAPID COMMUNICATION: Isolation of glucose transporters and in bovine uteroplacental tissues from days 16 to 50 of gestation.

Glucose transporter solute carrier family 2 member 14 () is a duplicon of glucose transporter solute carrier family 2 member 3 () with a 95% shared homology to and has not previously been isolated in ruminant uteroplacental tissues. The transporter has been previously isolated in Holstein heifer uterine epithelium but not in ovine epithelium. We hypothesized that and its duplicon would be found in bovine uteroplacental tissues and that maternal nutrition and day of gestation would impact mRNA expression of and . Crossbred Angus heifers ( = 49) were estrus synchronized, bred via AI, and assigned to nutritional treatment (CON = 100% of requirements to gain 0.45 kg/d; RES = 60% of CON) at breeding. Ovariohysterectomy was performed on d 16, 34, or 50 of gestation ( = 6 to 9/d); nonpregnant (NP) controls were not bred and ovariohysterectomized on d 16 of the synchronized estrous cycle ( = 6). The resulting treatment arrangement was a 2 × 3 factorial + 1. Uteroplacental tissues (caruncle, CAR; intercaruncular endometrium, ICAR; and fetal membrane [chorioallantois], FM) were obtained from the pregnant uterine horn immediately after ovariohysterectomy. For NP controls, only CAR and ICAR were obtained. There were no day × treatment interactions for or gene expression in CAR, ICAR, or FM. Expression of in CAR was greater ( = 0.03) on d 50 compared with d 16. In ICAR, was greatest ( = 0.02) on d 50 compared with d 16 and 34 of gestation. In FM, was greater ( = 0.04) on d 16 compared with d 50. Expression of was greater ( = 0.05) in pregnant compared with nonpregnant heifers. Additionally, expression of was greater ( = 0.01) on d 34 and 50 compared with d 16. Expression of in CAR was greater ( = 0.03) on d 50 compared to d 16 and 34. In CAR, tended ( = 0.07) to be greater on d 34 and 50 than on d 16 and was greater ( = 0.02) on d 50 than on d 34. There was no effect of treatment for either or in CAR, ICAR, or FM. These data demonstrate that glucose transporters and are expressed in beef heifer uteroplacental tissues and that they are expressed differentially by day of gestation in bovine uteroplacental tissues.

RAPID COMMUNICATION: Expression of an endogenous retroviral element, during early gestation in beef heifers.

Endogenous retroviral gene elements have been implicated in development and formation of the feto-maternal interface. A variant of the syncytin endogenous retroviral envelope gene family, , was recently found in ruminants. We hypothesized that mRNA would be differentially expressed in utero-placental tissues and would fluctuate during key time points of early gestation in beef heifers. Commercial Angus crossbred heifers ( = 46; ∼15 mo of age; BW = 362.3 ± 34.7kg) housed in 6-animal pens were fed daily with native grass hay and supplemented with cracked corn to gain 0.3 kg/d. The heifers were estrus synchronized, artificially inseminated, (d of breeding= d 0) and ovariohysterectomized on d 16, 22, 28, 34, 40, and 50 ( = 9, 6, 6, 7, 6, and 5, respectively) of gestation and at d 16 of the estrous cycle for non-bred, non-pregnant controls (NP; = 7). Harvested tissues were separated into maternal caruncle (CAR), intercarunclar endometrium (ICAR), and fetal membranes, (FM; chorioallantois, d 22 and later). All tissues were obtained from the ipsilateral uterine horn to the CL. Statistical analyses were conducted via the GLM procedure of SAS. Maternal CAR expression of was greater ( = 0.003) on d 50 by 81.5-fold compared to NP controls. At d 50 expression of in CAR was 190.3-fold greater than ( < 0.0001) ICAR. Fetal membranes had greater ( < 0.002) expression of from d 22 until d 50 of gestation compared to maternal ICAR (d 16 not analyzed). Expression of in FM was greater ( < 0.004) than in CAR until d 40 of gestation. Therefore, we conclude that is differentially expressed in utero-placental tissues and may be involved in the establishment of pregnancy. The expression of in maternal tissues is completely novel and indicates unique functions of syncytin in ruminant pregnancy.

Nutrient transporters in bovine uteroplacental tissues on days sixteen to fifty of gestation.

During early gestation, nutrients are transported to the developing embryo via transporters in the uterine endometrium and chorioallantois. In the present study, we examined glucose transporters and and the cationic AA transporters , , and to test the hypotheses that 1) relative mRNA expression of transporters would be different among uteroplacental tissue type as gestation progresses and 2) concentrations of glucose and cationic AA would be different among target sites (placental compartments, serum, and histotrophic) and days of gestation. To test these hypotheses, crossbred Angus heifers ( = 46) were synchronized, bred via AI, and then ovariohysterectomized on d 16, 22, 28, 34, 40, or 50 of gestation (5 to 9/d) or not bred and ovariohysterectomized on d 16 of the synchronized estrous cycle ( = 7) to serve as nonpregnant (NP) controls. Uteroplacental tissues (maternal caruncle [CAR], intercaruncular endometrium [ICAR], and fetal membranes [FM; chorioallantois, d 22 and later]) were collected from the uterine horn ipsilateral to the corpus luteum immediately following ovariohysterectomy. Relative mRNA expression of the glucose transporters and cationic AA transporters was determined for each tissue from d 16 to 50 of gestation and from NP controls. Chorioallantoic, amniotic, and plasma fluids were collected from heifers on d 40 and 50 of gestation to determine concentrations of glucose and cationic AA. Expression of and showed a tendency ( < 0.10) toward being greater in d 16 ICAR and d 34 ICAR, respectively. Day × tissue interactions ( < 0.05) were present for , , and . Expression of was greater in d 50 CAR, expression of was greater on d 34 in ICAR, and expression of was greater in CAR tissue on d 34 compared with all other tissues and days of gestation. Glucose concentrations tended ( = 0.10) to be impacted by a day × fluid interaction. A day × fluid interaction ( = 0.01) for arginine concentration was observed, with greater concentrations in allantoic fluid on d 40 compared with all other days and fluid types. These data support our hypothesis that glucose and cationic AA transporters differ in their level of mRNA expression due to day of gestation and uteroplacental tissue type. In addition, concentrations of nutrients were differentially impacted by day, target site, and/or their respective interaction.