Cathepsin-L Secreted by High-Quality Bovine Embryos Exerts an Embryotrophic Effect In Vitro

Abstract

While human in vitro embryo production is generally performed individually, animal models have shown that culturing embryos in groups improves blastocyst yield and quality. Paracrine embryotrophins could be responsible for this improved embryo development, but their identity remains largely unknown. We hypothesize that supplementation of embryotrophic proteins to a culture medium could be the key to improve individual embryo production. In this study, proteomics screening of culture media conditioned by bovine embryos revealed cathepsin-L as being secreted by both excellent- and good-quality embryos, while being absent in the medium conditioned by poor-quality embryos. The embryotrophic role of cathepsin-L was explored in vitro, whereby bovine zygotes were cultured individually for 8 days with or without cathepsin-L. Preliminary dose-response experiments pointed out 100 ng/mL as the optimal concentration of cathepsin-L in embryo culture medium. Supplementation of cathepsin-L to individual culture systems significantly improved blastocyst development and quality in terms of blastocoel formation at day 7, and the hatching ratio and apoptotic cell ratio at day 8, compared to the control. Taken together, cathepsin-L acts as an important embryotrophin by increasing embryo quality, and regulating blastulation and hatching in bovine in vitro embryo production.

Keywords: embryonic development; embryotrophins; in vitro culture.

Figure 1

Prevalence of membrane-damaged cells (MDCs) and total cell number (TCN) in bovine embryos, at day 8 post-fertilization. Embryos were cultured individually in droplets of 20 µL albumin-free culture medium and at day 8, a morphological evaluation of the blastocyst stage was performed using a stereomicroscope. Subsequently, all non-fixed viable embryos were subjected to Hoechst/propidium iodide staining to assess the TCN and the percentage of MDC. The boxplots show the median values (transition dark grey–light grey), 25th and 75th percentile (boxes), and 5th and 95th percentile (whiskers). All embryos (n = 606) were collected from four replicates, the number of embryos belonging to a developmental stage (YB = young blastocyst; NB = normal blastocyst; EB = expanded blastocyst; HB = hatching or hatched blastocyst) is represented in the figure.

Figure 2

Venn diagram depicting all protein accessions identified in the compared media after subtraction of the proteins in the blank medium. Compared media: ‘MS Run 1: Good and Poor’: pooled medium conditioned by good- and poor-quality embryos, respectively; ‘MS Run 2: Excellent and Poor’: pooled medium conditioned by excellent- and poor-quality embryos, respectively. Numbers from the Venn diagram are depicted with their respective Uniprot accession numbers in the periphery. Protein descriptions can be found in Supplementary Tables S1 and S2. Cathepsin-L (CATL) was the only protein exclusively identified in ‘MS Run 1: Good’ and ‘MS Run 2: Excellent’.

Figure 3

Visual representation of the research described in the present manuscript. 1. Identification of paracrine embryotrophins in embryo-conditioned medium: (1.1) Embryo and medium collection: individual culture of bovine embryos in albumin-free SOF/PVP/ITS medium. Droplets of SOF/PVP/ITS medium without embryo were incubated under the same conditions and served as a blank control. At day 8 post-fertilization, each embryo, droplets of EC medium and droplets of blank medium were collected separately. (1.2) Determination of TCN and MDC ratio: all embryos were subjected to a Hoechst/propidium iodide staining to differentiate between membrane intact and MDCs. Based on the results, embryo quality was categorized as ‘excellent’, ‘good’ or ‘poor’ quality in this context. (1.3) Protein identification: EC medium of good-quality embryos was pooled and compared to pooled EC medium of an equivalent number of poor-quality embryos and an equivalent volume of blank droplets in MS run 1. Under similar conditions, pooled EC medium of excellent-quality embryos, pooled EC medium of poor-quality embryos and blank medium were compared in MS run 2. 2. Confirmation of the embryotrophic effect of cathepsin-L. (2.1) In vitro embryo production: individual culture of bovine embryos in the control medium was compared to individual culture in cathepsin-L-supplemented medium (100 ng/mL). Embryos cultured in a group in the control medium were included as an extra control group. (2.2) Differential apoptotic staining: blastocysts collected at day 8 post-fertilization from exp 2.1 were subjected to differential apoptotic staining. Abbreviations: EC, embryo conditioned; ITS, insulin transferrin selenium; MDC, membrane-damaged cell; MS, mass spectrometry; PVP, polyvinylpyrrolidone; SOF, synthetic oviductal fluid; TCN, total cell number.

Figure 4

Brightfield (A) and fluorescent (B–E) images of differential apoptotic staining in a bovine blastocyst. At 8 days post-fertilization, bovine blastocysts were fixed and immuno-stained for CDX2 (red), (B) and active caspase-3 (green), (C). Similarly, Hoechst staining was performed to stain all nuclei (blue), (D). The merged image (E) shows the distribution of the different cell types in the blastocyst. The image was acquired by confocal microscopy using a 20x objective and represents a hatching blastocyst derived from the control group.