128 Evidence that Pregnancy-Associated Serum Protein A (PAPP-A) Plays Role on Bovine In Vitro Embryo Production

Abstract

The aim of present work was to assess the effects of pregnancy-associated serum protein A (PAPP-A) during oocyte in vitro maturation (IVM) on meiosis progression, DNA fragmentation, IGF-1 free bioavailability, as well as effects on embryo yield and transcriptional profile of matured cumulus–oocyte complexes (COC). First, the COC from a local abattoir were submitted to IVM for 24 h with TCM-199 serum-free medium supplemented with PAPP-A (100 ng mL⁻¹: P100 group) or not (control group). The matured oocytes were submitted to evaluation of DNA fragmentation (TUNEL assay) and meiosis progression (Hoechst 33342; n = 5 replicates; 20 COC/replicate per group), and maturation medium was collected to measure levels of free IGF-1. Then, the oocytes were separated from their respective cumulus cells and followed for the transcriptional profile of 96 genes (3 reference genes; ACTH, GAPDH, PPIA) by RT-qPCR using Taqman^® assays in the HD-Biomark System^® (Fluidigm Corp., South San Francisco, CA, USA). Further, the matured oocytes were submitted to in vitro fertilization followed by in vitro culture for 7 days. On Days 3 and 7, the cleavage and blastocyst (BL) rates were verified. On Day 7, BL (3 BL/pool; control: n = 4 pools; P100: n = 5 pools) were collected to analyse the transcriptional pattern of 96 genes (4 reference genes; ACTH, GAPDH, PPIA, and SDHA) as described above for COC. The DNA fragmentation, meiosis progression, cleavage, and BL rates were calculated as percentages and transformed to arcsine. The mRNA abundance of target genes was normalized by geometric mean of reference genes and data were transformed to fold change. The free IGF-I concentration also was transformed to fold change. All data were tested by ANOVA and means were compared with t-test or Wilcoxon tests using JMP software (SAS Institute Inc., Cary, NC, USA). Differences were considered significant when P ≤ 0.05. The addition of PAPP-A increased free IGF-I concentration 1.27-fold in IVM medium. There were no alterations in the percentage of oocytes in metaphase II or oocyte DNA fragmentation. In cumulus cells, the genes BCL2, GPX1, RPLP0, and RPS25 (anti-apoptotic and anti-oxidative stress) was higher in the P100 group, whereas DICER, GREM1, GUCY1B3, and FOXO3 (cell proliferation, cumulus expansion, cGMP regulator, and apoptotic initiator, respectively) were higher in the control group. In oocytes, the mRNA relative abundance of ACACA, BCL2, H1FOO, TXNRD1, and VCAN (related with fatty acid synthesis, anti-apoptotic effect, chromatin regulation, oxidative stress processes, and cell proliferation, respectively) was higher in the P100 group. There was no difference in cleavage rate or embryo yield. The mRNA abundance of genes related to cellular stress (ATF4, GPX4, and HIF1A) and lipid metabolism (FASN and SREBF1) was lower in embryos of the P100 group. On the other hand, genes involved in cellular proliferation/differentiation (MAPK1) and pluripotency (POU5F1) were up-regulated in embryos of the P100 group. In conclusion, the addition of PAPP-A during the IVM increased free IGF-I and modulates the gene expression in COC and blastocysts, which could modify oocyte competence and embryo development.