Supplementing in vitro embryo production media by NPPC and sildenafil affect the cytoplasmic lipid content and gene expression of bovine cumulus-oocyte complexes and embryos.

In our study, we added natriuretic peptide type C (NPPC) and/or sildenafil during in vitro maturation (IVM) of bovine cumulus-oocyte complexes (COCs) followed by in vitro culture (IVC) of embryos with or without sildenafil. We evaluated the effects on the lipid content (LC) of oocytes and embryos and also verified the expression of 96 transcripts related to competence in matured COCs and 96 transcripts related to embryo quality in blastocysts. After IVM, LC was decreased in oocytes by NPPC while sildenafil did not affect LC in oocytes. The genes involved in lipid metabolism and lipid accumulation (DGAT1, PLIN2and PLIN3) were not affected in COCs after treatment during IVM, although the expression of PTX3 (a cumulus cells expansion biomarker) was increased and the hatched blastocyst rate was increased by NPPC during IVM. During IVM, sildenafil increased the mRNA relative abundance of HSF1 and PAF1 and decreased REST in blastocysts. The use of sildenafil in IVC increased the LC of blastocysts. The mRNA abundance in blastocysts produced during IVC with sildenafil was changed for ATF4, XBP1, DNMT3A, DNMT3B, COX2, and SOX2. Although NPPC reduced the LC of oocytes after IVM and upregulated markers for cumulus expansion, embryo production was not affected and the produced blastocysts were able to regain their LC after IVC. Finally, the use of sildenafil during IVC increased the cytoplasmic LC of embryos but did not affect embryo quality, as measured by analysis of 96 transcripts related to embryo quality.

Cell apoptosis and lipid content of in vitro-produced, vitrified bovine embryos treated with forskolin.

The presence of fetal calf serum in culture medium influences embryo quality, causing a reduction in postcryopreservation survival. Forskolin has been used to induce lipolysis and increase cryotolerance, functioning as an activator of adenylate cyclase and elevating cAMP levels. In the present experiment, bovine zygotes were cultured in synthetic oviduct fluid with amino acid plus 2.5% fetal calf serum for 6 days, when forskolin was added in three concentrations: 2.5, 5, and 10 µM. Treatment with forskolin lasted for 24 hours. Blastocyst formation rate, quantification of lipid granules, total cell numbers, and apoptosis rate were evaluated. In a second assessment, embryos were vitrified, and warming, re-expansion rate, total cell numbers, and apoptosis rate were also evaluated. There was no difference due to forskolin in blastocyst formation or re-expansion rates after vitrification. However, lipid measurements were lower (control: 136.8 and F 2.5 µM: 128.5; P < 0.05), and number of cells per embryo higher (control: 140.1 and F 2.5 µM: 173.5; P < 0.05) than controls for 2.5 µM forskolin but not for higher forskolin concentrations. The number of intact cells per embryo was higher, and the rate of apoptosis was lower in fresh than in vitrified embryos (number of cells of warmed embryos, control: 104.1, F 2.5 µM: 101.3, F 5 µM: 115.4, F 10 µM: 95.1; apoptotic of fresh cells, control: 12.1%, F 2.5 µM: 16.7%, F 5 µM: 11.1%, F 10 µM: 14.2%; and apoptotic warmed embryos, control: 22.3%, F 2.5 µM: 37.3%, F 5 µM: 33.2%, F 10 µM: 30.3%; P < 0.05). It was concluded that forskolin is an effective lipolytic agent even at low concentrations, leading to formation of blastocysts with a comparatively larger number of cells.