Wang X, Dunlap K, Satterfield M, Frank J, Li X, Ying W, Burghardt R, Johnson GA, Wu G, Antoniazzi AQ, Nett TM, Hansen TR, Bazer FW

Arginine (Arg), a nutritionally essential amino acid for conceptus development, can be metabolized to nitric oxide (NO) and polyamines. Arg increases 13-fold in the ovine uterine lumen between Days 10 and 15 of pregnancy and activates mechanistic target of rapamycin cell signaling. This study tested the hypothesis that knockdown of translation of Arg-associated gene transcripts inhibits ovine conceptus development. Morpholino antisense oligonucleotides (MAOs) were designed to inhibit translation of mRNAs for: 1) SLC7A1, Arg transporter; 2) NOS3, an enzyme for NO synthesis; 3) ODC1, an enzyme for polyamine synthesis; and 4) mismatch morpholino as MAO-control. On Day 8 post-mating, the uterine horn of ewes (n=7 or 8 /MAO type) ipsilateral to the CL was ligated and MAO injected into the uterine lumen. On Day 16, ewes were hysterectomized to obtain conceptuses, uterine flushings, and uterine tissues from the treatment groups: 1) MAO-SLC7A1 (n=8); 2) MAO-NOS3 (n=7); 3) MAO-ODC1 (n=8); and 4) MAO-control (n=7). Conceptuses in the control uterine horn were healthy, elongated and intact. Conceptuses exposed to the SLC7A1-MAO were small, thin, fragile and fragmented. Conceptuses exposed to the NOS3-MAO were thin and small, but elongated and healthy. In the ODC1-MAO group, half of the conceptus were small, fragile and fragmented, but half were healthy and elongated. Compared to protein concentration (2.1 + 0.5 mg/ml), uterine flush volume recovered (8.2 + 0.8 ml) and total protein in uterine flushing (15.7±3.5 mg) for MAO-control ewes, values did not differ (P>0.05) for treatment MAOs. Slot blot analysis did not detect effects of treatment (P>0.05) on interferon-tau (IFNT) in uterine flushings. However, radioimmunoassay data revealed an increase (P<0.05) in IFNT in MAO-NOS3 (15,057±2,538 ng) compared to MAO-control (9,353±1,768 ng) ewes. Pregnancy rates were not affected by treatment (P>0.2). Thus, knockdown of SLC7A1 protein attenuated conceptus development. Knockdown of NOS3 and ODC1 proteins partially suppressed conceptus development likely due to compensatory effects of NOS1/NOS2, other cationic amino acid transporters and production of putrescine via the arginine decarboxylase and agmatinase pathway.