Proteomic Analysis of Domestic Cat Blastocysts and Their Secretome Produced in an In Vitro Culture System without the Presence of the Zona Pellucida

Abstract

Domestic cat blastocysts cultured without the zona pellucida exhibit reduced implantation capacity. However, the protein expression profile has not been evaluated in these embryos. The objective of this study was to evaluate the protein expression profile of domestic cat blastocysts cultured without the zona pellucida. Two experimental groups were generated: (1) domestic cat embryos generated by IVF and cultured in vitro (zona intact, (ZI)) and (2) domestic cat embryos cultured in vitro without the zona pellucida (zona-free (ZF group)). The cleavage, morula, and blastocyst rates were estimated at days 2, 5 and 7, respectively. Day 7 blastocysts and their culture media were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS). The UniProt Felis catus database was used to identify the standard proteome. No significant differences were found in the cleavage, morula, or blastocyst rates between the ZI and ZF groups (p > 0.05). Proteomic analysis revealed 22 upregulated and 20 downregulated proteins in the ZF blastocysts. Furthermore, 14 proteins involved in embryo development and implantation were present exclusively in the culture medium of the ZI blastocysts. In conclusion, embryo culture without the zona pellucida did not affect in vitro development, but altered the protein expression profile and release of domestic cat blastocysts.

Keywords: embryo–maternal communication; felid embryos; in vitro fertilization; protein expression profile; zona pellucida.

Figure 1

Domestic cat blastocysts. (A) Domestic cat blastocyst from the ZI group at day 7 of IVC (20X). (B) Domestic cat ZI blastocyst stained with Hoechst (20X). (C) ZI blastocyst merge (Hoechst and transmitted light) (20X). (D) Domestic cat blastocyst from the ZF group at day 7 of IVC (20X). (E) Domestic cat ZF blastocyst stained with Hoechst (20X). (F) ZF blastocyst merge (Hoechst and transmitted light) (20X).

Figure 2

Morphological evaluation of blastocysts. (A) Total cell number in blastocysts (mean; min/max) from the ZI (206.0; 122/289) and ZF groups (219.4; 111/377). (B) Diameter (mean; min/max) of blastocysts from the ZI (241.2; 142.9/444.8 µm) and ZF groups (304.4; 149.8/520.3 µm). * Significant differences between bars, p < 0.05.

Figure 3

Venn diagrams of the proteins identified in the ZF and ZI blastocysts. (A) Comparison among the protein groups identified in blastocyst samples: ZF01, ZF02 and ZF03 (ZF group). (B) Comparison among the protein groups identified in blastocyst samples: ZI01, ZI02 and ZI03 (ZI group). (C) Comparison between the total proteins identified in the ZF and ZI groups. In sum, 456 proteins were shared between groups: 1148 were exclusively detected in ZF blastocysts and 26 were expressed only by ZI blastocysts.

Figure 4

Heatmap of DEPs identified in the blastocysts (ZF vs. ZI). The 42 DEPs are grouped into blocks. Each block corresponds to proteins with similar expression patterns.

Figure 5

Volcano plot of the DEPs (ZF vs. ZI). In sum, 250 proteins were quantified: 22 were upregulated and 20 were downregulated (FDR < 0.05).

Figure 6

Functional classification (descriptive analysis) of DEPs identified between ZF vs. ZI blastocysts. Pie charts were created based on the functional classification of the PANTHER18.0 classification system, UniProt GO annotation, and QuickGO ancestor chart. Upregulated proteins (A–C). (A) Molecular function (GO:0003674), (B) biological process (GO:0008150), and (C) cellular component (GO:0005575). Downregulated proteins (D–F). (D) Molecular function (GO:0003674), (E) biological process (GO:0008150), and (F) cellular component (GO:0005575).

Figure 7

Venn diagram of proteins identified in the conditioned culture medium of blastocysts: 25 proteins were shared between ZF and ZI samples, 5 proteins were detected only in the culture medium of ZF blastocysts, and 14 in the culture medium of ZI blastocysts.

Figure 8

Functional classification (descriptive analysis) of proteins detected in the conditioned culture media of ZF and ZI blastocysts. Pie charts were created based on the functional classification of the PANTHER18.0 classification system, UniProt GO annotation, and QuickGO ancestor chart. (A–C) Proteins identified in the conditioned culture medium of ZF blastocysts. (D–F) Proteins identified in the conditioned culture medium of ZI blastocysts. (G–I) Proteins shared by the samples of conditioned culture media of ZF and ZI blastocysts.

Figure 9

Experimental design. Workflow diagram of in vitro fertilization, embryo culture and proteomic analysis of day 7 blastocysts (experiment 1) and their conditioned culture media (experiment 2).