Effects of Cryptorchidism on the Semen Quality of Giant Pandas from the Perspective of Seminal Plasma Proteomics

Abstract

Giant pandas are an endangered species with low reproductive rates. Cryptorchidism, which can negatively affect reproduction, is also often found in pandas. Seminal plasma plays a crucial role in sperm-environment interactions, and its properties are closely linked to conception potential in both natural and assisted reproduction. The research sought to identify seminal fluid protein content variations between normal and cryptorchid giant pandas. Methods: Using a label-free MS-based method, the semen proteomes of one panda with cryptorchidism and three normal pandas were studied, and the identified proteins were compared and functionally analyzed. Results: Mass spectrometry identified 2059 seminal plasma proteins, with 361 differentially expressed proteins (DEPs). Gene ontology (GO) analysis revealed that these DEPs are mainly involved in the phosphate-containing compound metabolic, hydrolase activity, and kinase activity areas (p ≤ 0.05). The KEGG functional enrichment analysis revealed that the top 20 pathways were notably concentrated in the adipocyte lipolysis and insulin metabolism pathway, with a significance level of p ≤ 0.05. Further analysis through a protein-protein interaction (PPI) network identified nine key proteins that may play crucial roles, including D2GXH8 (hexokinase Fragment), D2HSQ6 (protein tyrosine phosphatase), and G1LHZ6 (Calmodulin 2). Conclusions: We suspect that the high abundance of D2HSQ6 in cryptorchid individuals is associated with metabolic pathways, especially the insulin signal pathway, as a typical proteomic feature related to its pathological features. These findings offer insight into the ex situ breeding conditions of this threatened species.

Keywords: cryptorchidism; giant panda; proteomics; seminal plasma.

Figure 1

Statistics of the identified seminal plasma proteins in the giant panda. (A) Sample grouping statistics from tandem mass spectrometry and protein identification. (B) Functional annotations of proteins in public databases.

Figure 2

Functional annotation of the identified seminal plasma proteins in giant panda semen. (A) Subcellular localization of the identified proteins. (B) GO enrichment analysis of the identified proteins. (C) IPR analysis of the identified protein. (D) KEGG analysis of the identified proteins.

Figure 3

Principal component analysis (PCA) and GO terms and KEGG pathway enrichment DEPs analysis of seminal plasma proteins in cryptorchid and normal testis semen samples. (A) PCA of cryptorchid and normal testis semen sample. According to the distribution of PC1 and PC2, the normal testes group were divided into ellipses. PC1. (B) Volcano diagram of DEPs between cryptorchid and normal testis group. (C) Hierarchical clustering of DEPs. The vertical clustering is the sample clustering, and the horizontal clustering is the protein clustering. Enriched GO terms (D), top 20 enriched KEGG pathways (E), enrichment of DEPs.

Figure 4

KEGG pathway enriches upregulated and downregulated DEPs between the cryptorchid and the normal testis group. (A) The first 20 enriched upregulated KEGG pathways. (B) The first 20 enriched downregulated KEGG pathways. The Abscissa in the picture is the ratio of the number of differential proteins in the corresponding pathway to the total number of proteins identified by the pathway. The higher the ratio, the higher the degree of enrichment of differential proteins in this pathway.

Figure 5

Network analyses of DEPs between the cryptorchid and normal testis groups. (A) PPI enrichment analysis showing physical interactions formed among DEPs in the cryptorchid and normal testis groups. (B) Insulin signal pathway map involving cryptorchidism sexual characteristics related to seminal plasma proteins.