91 Haptoglobin addition during in vitro culture influences gene expression related to oxidative stress and lipid metabolism in bovine embryos

Haptoglobin (HG) is a liver-synthesized protein commonly associated with inflammation, but it is also present in healthy mammalian reproductive tissues and fluids. In a previous study, we showed that exogenous HG significantly enhanced in vitro bovine blastocyst development. Here, we aimed to evaluate the effect of HG on the quality of in vitro bovine embryos and to explore the gene expression associated with cell proliferation and embryo quality. Zygotes were cultured in 25 µL of in vitro culture medium alone (Stroebech Media®; Control, n = 516) or supplemented with 5 μg of HG (H5, n = 519) during the entire culture period (Days 1–7 [D1–D7]) or during two developmental periods: (1) D1–D4, from presumptive zygotes to 16-cell stage (representing HG effect in the oviduct; H5-C, n = 446), or (2) D4–D7: from 16-cell stage to blastocyst (BD7; representing HG effect in the uterus; C-H5, n = 438). For differential staining analysis, BD7 from each group were incubated with anti-CDX2 antibody (Biogenix) and stained with Hoechst (20/group). Additionally, on D7, three pools of 10 BD7 were snap-frozen in liquid nitrogen and stored at −80°C until further analyses. The selected genes have been linked to cell proliferation and embryo quality, such as POU5F1, CDK2, CHD1, PPARGC1B, SOD1, PLIN2, GPX1, NFE2L2, and BAX. H2AFZ and ACTB were used as housekeeping genes. For immunofluorescence analysis, BD7 (15/group) from all experimental groups were incubated with PLIN2 (Santa Cruz Biotechnology) and SOD1 (ThermoFisher) antibodies. All BD7 were examined with a confocal microscope, and images were evaluated with ImageJ program. Data were analyzed by one-way ANOVA followed by the Tukey test. Total cell, trophectoderm, and inner cell mass did not show differences in BD7 cultured with H5 (117.1 ± 0.9, 76.6 ± 0.9, and 40.5 ± 0.7, respectively) during the entire culture period (D1–D7) compared with D1–D4 (H5-C: 117.2 ± 0.9, 75.7 ± 0.7, and 41.5 ± 0.5, respectively), D4-D7 period (C-H5: 117.7 ± 1.0; 76.2 ± 1.0; 41.4 ± 0.8, respectively), or without HG (control: 118.6 ± 1.1, 76.9 ± 0.9, and 41.0 ± 0.7, respectively; P > 0.05). The expression level of the SOD1 gene was significantly increased in blastocysts from the H5 and C-H5 groups compared with the control and H5-C groups. While the expression of the PLIN2 gene was higher in the control and H5-C groups compared with the H5 and C-H5 groups (P < 0.05), the CDK2 gene was upregulated in all HG groups compared with the control (P < 0.05). No significant differences were observed for NFE2L2, PPARGC1B, POU5F1, CDH1, GPX1, and BAX transcripts. Immunofluorescence analysis revealed that SOD1 increased its fluorescence levels in H5 or during C-H5, while PLIN2 levels were weaker in blastocysts from H5 or during C-H5. In conclusion, these findings highlight the potential benefits of HG addition in improving embryo quality. HG appears to influence embryonic development by modulating gene expression and protein levels in blastocysts, suggesting roles in reducing oxidative stress and lipid content and regulating developmental pathways.

This study was supported by a grant from Maria Zambrano (KCB); Fundación Margarita Salas (YNC); Spanish Ministry of Science and Innovation: PID2019–111641RB-I00 and PID2021-123091NBC21: MCIN/AEI /10.13039/501100011033/ and FEDER Una manera de hacer Europa.