58 The embryotrophic effect of cathepsin-L in a bovine in vitro model

While human in vitro embryo production is generally performed individually, animal models have shown that culturing embryos in groups significantly improves blastocyst quality. Paracrine embryotrophins could be responsible for the improved embryo development in groups, but their identity remains largely unknown. Since this paracrine effect is absent in the individual system, we hypothesise that supplementation of embryotrophins to the culture medium might be the key for successful individual embryo production. Proteomics screening of culture media conditioned by bovine embryos revealed cathepsin-L as the only protein that was secreted by embryos of excellent and good quality. Known to be involved in early stem cell differentiation and embryo-maternal interactions in multiple species including humans, the embryotrophic properties of cathepsin-L were further explored in a bovine model. To this end, immature bovine oocytes were harvested from slaughterhouse ovaries for in vitro embryo production. Oocytes were cultured for 22 h in maturation medium (TCM199 + 15 µg mL⁻¹ gentamycin supplemented with 20 ng mL⁻¹ epidermal growth factor), followed by co-incubation with 1 × 10⁶ spermatozoa mL⁻¹ for 21 h in fertilisation medium (Tyrode’s albumin lactate pyruvate supplemented with bovine serum albumin and heparin). Presumed zygotes were then transferred to synthetic oviducal fluid (enriched with bovine serum albumin, insulin, transferrin, and selenium), with or without cathepsin-L at a concentration of 100 ng mL⁻¹ and incubated at 38.5°C in 5% CO₂, 5% O₂, and 90% N₂ for 8 days. In total, 3003 presumed zygotes were cultured in six replicates. In vitro maturation and fertilisation were performed in groups (60 oocytes/500 µL), while embryo culture was done individually (1 presumed zygote/20 µL). Consequently, a fluorescent differential staining was applied on Day 8 blastocysts to count all cells (Hoechst), trophoblast cells (CDX-2 antibody) and apoptotic cells (anti-caspase antibody). All data were analysed in RStudio. Developmental rates were analysed using generalised linear mixed-effect models with replicate nested as a random effect. Cell counts were checked for normality by quantile-quantile plots and Shapiro-Wilk tests and analysed by one-way ANOVA or Kruskal-Wallis tests, depending on data distribution. Supplementation of cathepsin at 100 ng mL⁻¹ resulted in an increased blastocyst rate (i.e. the total number of blastocysts/total number of fertilised oocytes) at Day 7 (27.6 ± 1.8% vs 20.9 ± 1.6%; P = 0.0196), an increased hatching rate (i.e. the total number of hatching or hatched blastocysts/total number of blastocysts) at Day 8 (19.9 ± 2.7% vs 7.7 ± 1.8%; P = 0.0008), and a lower number of apoptotic cells (4 ± 2 vs 5 ± 4; P = 0.016) and reduced apoptotic cell ratio (3.1 ± 2.9% vs 4.4 ± 3.3%; P = 0.045) compared with the control group. Taken together, we identified cathepsin-L as an important embryotrophin in a bovine model. Cathepsin-L improved blastocyst development and quality in terms of hatching rate and apoptotic cell ratio. Whether this protein also plays a role in human embryo development remains to be determined.