Result
A total of 1125 proteins in the sperm plasma (Table S1, Supporting Information) were identified using the label-free shotgun proteomic approach. Researchers can understand the functional characteristics of different proteins by utilizing these databases to perform functional annotations on identified proteins. In this study, we used four databases (GO, IPR, KEGG and COG) for annotation, most proteins could be annotated (Figure 1A). , Based on the results of the GO annotation analysis of the cellular compartment, there is a broad range of sources for seminal plasma proteins, and the primary sources are intracellular, ribosome and membrane (Figure 1B). The molecular function of these proteins is associated with protein binding, ATP binding, calcium ion binding and so on, they are mainly involved in the oxidation-reduction process, proteolysis, translation and carbohydrate metabolic process. We found that these proteins are rich in EF-hand domain, intermediate filament and immunoglobulin-like domain (Figure 1C), which should match their functions. Based on COG and KEGG annotation (Figure 2), it also highlights that these proteins are involved in protein turnover, translation, and metabolism, which may play a major role in the immune and endocrine systems.
As different species may use different versions of proteins, gene names corresponding to proteins were used uniformly for comparative analysis. A comparison of the seminal plasma proteins identified in giant pandas and three other species (human(Saraswat et al., 2017), pig(Perez-Patino et al., 2016), sheep(Soleilhavoup et al., 2014) ) revealed that pandas share 25 genes (corresponding to 25 proteins) with the other species, and have 598 unique genes (corresponding to 598 proteins). Subsequent functional enrichment analysis shows that these shared proteins mainly play a role in the ion binding process (Figure 3A), seminal plasma contains a variety of ions including sodium, potassium, calcium, magnesium, and zinc(Sorensen et al., 1999), and the ion binding process is important for the regulation of the pH and osmotic pressure of the semen, as well as for the function and viability of sperm. The COG annotated these proteins in serum albumin and fibronectin, human serum albumin (HSA) is the most abundant seminal plasma protein and an important constituent of seminal plasma (Figure 3B), it has an established role as a sink for cholesterol and is removed from the sperm membrane during capacitation(Kumar et al., 2012), albumin is believed to play a role in the maintenance of sperm motility(Mogielnicka-Brzozowska et al., 2019), and may also have antioxidant properties that protect the sperm from oxidative stress. Fibronectin is a ubiquitous multifunctional glycoprotein and a component of the seminal fluid. It plays a key role in the formation of seminal gel following ejaculation and can bind to cellular components that are exposed when a spermatozoon is damaged and thus helps select abnormal spermatozoa. Overall, both albumin and fibronectin are important components of seminal plasma and may play important roles in the function and viability of sperm. Additionally, researchers are investigating these proteins’ potential diagnostic and therapeutic uses in the context of male infertility and other reproductive disorders.
In the comparative analysis, most proteins were unique to giant pandas, which may be related to the unclear annotation of proteins in different species. Functional analysis was also conducted on panda unique proteins, these proteins were enriched in the ribosome pathway (Figure 3C), and were also related to binding function, including heterocyclic compound binding and nucleic acid binding (Figure 3D). As we know, sperm do not have ribosomes and hence do not synthesize proteins, during sperm development, ribosomes are present in the early stages, but they are degraded and eliminated as the sperm matures. Therefore, instead of synthesizing proteins, sperm rely on the proteins that are produced by the cells of the male reproductive system and are present in the seminal fluid. Additionally, these proteins were associated with WD40 repeat and WD40 repeat-containing domains (Figure 3E). The WD40 repeat is a short motif structure consisting of approximately 40 amino acids that are often in a tryptophan-aspartic acid (W-D) dipeptide(Neer et al., 1994). The WD40 domain often comprises several of these repeats and is found in many proteins that are involved in a variety of cellular processes(Li & Roberts, 2001). Previous research has shown that the WD40 repeat containing proteins, including DDB1-CUL4-associated factors (DCAFs), are abundant and conserved proteins that play important roles in different cellular processes, including spermatogenesis (Mistry et al., 2020). Several proteins containing WD40 repeats have been identified in seminal plasma, including beta-catenin, which is involved in cell adhesion and signaling(Takezawa et al., 2011), and ring finger protein 17 (RNF17), which is a ubiquitin ligase that regulates protein degradation(Liu et al., 2011). These proteins are thought to play important roles in sperm function and fertility (Pan et al., 2005; Rivas et al., 2014), although the exact mechanisms are not yet fully understood. In humans, WDR62(Qin et al., 2019), WDR63(Lu et al., 2021), WDR66(Kherraf et al., 2018) have been identified and implicated in abnormalities of the sperm flagellum and male infertility. A recent study found a non-synonymous point mutation in a WD-40 domain repeat of EML5 leads to decreased bovine sperm quality and fertility(Nogueira et al., 2022).
In this study, twelve WD40 repeat-containing proteins were identified, we presented fundamental information about these repeats, and their molecular functions were not directly associated with sperm. Through a literature review, we discoveredPAFAH1B1plays important roles in spermatogenesis, fertilization and subsequent embryonic development in mice (Yao et al., 2015). We believe that these proteins in the seminal plasma are related to the formation and functional performance of sperm, but further research is still needed, especially for giant pandas.
Table 1 Summary of WD40 repeat-containing proteins in seminal plasma.