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.