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\begin{document}

\title{The seminal plasma proteome of the giant panda}



\author[1]{Kailai Cai}%
\author[2]{Tao Wang}%
\author[1]{Jiasong Chen}%
\author[1]{Feiping Li}%
\author[1]{Shenfei  Wang}%
\author[1]{Mengshi Zhang}%
\author[1]{Xianbiao Hu}%
\author[1]{Ayala James}%
\author[1]{Junhui An}%
\author[1]{Donghui Wang}%
\author[1]{Mingyue Zhang}%
\author[1]{Hairui Wang}%
\author[1]{He Huang}%
\author[1]{Yuliang Liu}%
\author[1]{Rong Hou}%
\affil[1]{Chengdu Research Base of Giant Panda Breeding}%
\affil[2]{Chengdu University}%


\vspace{-1em}



  
  \date{June 17, 2024}


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\selectlanguage{english}
\begin{abstract}
For the ex-situ conservation of giant pandas, both collecting and
preserving semen are important methods. The seminal plasma is rich in
nutrients and bioactive substances, such as proteins, carbohydrates,
lipids, amino acids, and hormones, which play an important role in the
reproduction and reproductive health of the species. This is the first
study to analyze the seminal plasma proteins of giant pandas through
proteomics and identified 1125 proteins. These proteins are related to
protein turnover, translation, and metabolism. The seminal plasma
proteins of giant pandas were then compared to those of humans, pigs and
sheep, with many unique proteins found in giant panda samples. Among
these proteins, the WD40 repeat-containing proteins have been identified
and implicated in sperm function and fertility. Understanding the
composition and function of proteins in the giant panda seminal plasma
proteome can provide valuable insights into their reproductive biology
and help develop strategies to improve their reproductive success in
captivity, which is essential for giant panda conservation.%
\end{abstract}%



\sloppy


The seminal plasma proteome of the giant panda

Abstract

For the \emph{ex-situ}conservation of giant pandas, both collecting and
preserving semen are important methods. The seminal plasma is rich in
nutrients and bioactive substances, such as proteins, carbohydrates,
lipids, amino acids, and hormones, which play an important role in the
reproduction and reproductive health of the species. This is the first
study to analyze the seminal plasma proteins of giant pandas through
proteomics and identified 1125 proteins. These proteins are related to
protein turnover, translation, and metabolism. The seminal plasma
proteins of giant pandas were then compared to those of humans, pigs and
sheep, with many unique proteins found in giant panda samples. Among
these proteins, the WD40 repeat-containing proteins have been identified
and implicated in sperm function and fertility. Understanding the
composition and function of proteins in the giant panda seminal plasma
proteome can provide valuable insights into their reproductive biology
and help develop strategies to improve their reproductive success in
captivity, which is essential for giant panda conservation.

KEYWORDS

Giant panda, seminal plasma, proteome, WD40 repeats proteins

The giant panda (\emph{Ailuropoda melanoleuca} ) is a unique and
vulnerable species endemic to China. It is not only a flagship species
for global biodiversity conservation, but also serves as a political and
diplomatic ambassador for China, and a cultural icon. Conserving the
giant panda is of particular ecological, social, political and economic
significance.

In recent years, both \emph{in-situ} and \emph{ex-situ} conservation
measures have been implemented to different degrees of success, however,
the species is still vulnerable to extinction. For the captive
population, the low proportion of naturally mating male giant pandas is
a major limiting factor for their reproductive efficiency, utilization,
and genetic diversity. With artificial insemination techniques, this
problem can be overcome and genetic management can also be facilitated,
making the preservation and efficient use of individual giant panda
semen significant for maintaining the entire captive population.

Seminal plasma is a fluid that is produced by the male reproductive
system and is a mixture of secretions from the testis, epididymis and
male accessory sex glands. Sperm motility is an index tightly associated
with male fertility(Jia et al., 2021). Seminal plasma proteins have
important roles in sperm functionality, and different mechanisms
including micro-vesicle transport of proteins are involved in the
regulation of sperm biology. Due to the role of seminal plasma, specific
proteins present in seminal plasma may be used as discriminant variables
with the potential to predict sperm motility and
fertility(Gaitskell-Phillips et al., 2022). The seminal plasma proteome
refers to the complete set of proteins in the fluid that makes up semen,
including enzymes, transport proteins, and immune system components. The
seminal plasma proteome contains thousands of proteins and includes many
tissue-specific proteins that might accurately indicate a pathological
process in the tissue of origin(Drabovich et al., 2014). Several seminal
plasma proteins are associated with male fertility but most of these
have not been studied in detail until now(Candenas \& Chianese, 2020;
Cannarella et al., 2020).

Research on the seminal plasma proteome has been ongoing for many years
and has yielded important insights into male reproductive biology and
the mechanisms underlying male infertility. Camargo, M. et al used
proteomic techniques to analyze the seminal plasma proteome of men with
and without a varicocele, a common cause of male infertility (Camargo et
al., 2013). The researchers identified several differentially expressed
proteins in the two groups, suggesting that these proteins may be
involved in the pathogenesis of varicocele-associated male infertility.
Another study used mass spectrometry to analyze the seminal plasma
proteome of fertile and infertile men(da Silva et al., 2016). The
researchers identified several proteins that were present at
significantly different levels in the two groups, suggesting that these
proteins may be useful biomarkers for male infertility. Recently,
Martins, A.D. et al elucidated the potential role of differentially
expressed proteins in the seminal plasma as a diagnostic biomarker for
primary and secondary infertility, their results showed overexpression
of ANXA2, CDC42 and under-expression of SEMG2 proteins in primary
infertility; and overexpression of ANXA2 and APP proteins in secondary
infertility(Martins et al., 2020). For instance, the correlation between
semen protein composition, sperm activity and fertility in animals such
as cattle(Westfalewicz et al., 2017), goats(Jia et al., 2021),
pigs(Mills et al., 2020) and chicken(Li et al., 2020) has been explored.

Zhu et al quantified 35 metabolome molecules with distinct age-related
trends in the giant panda seminal plasma(Zhu et al., 2022). However, the
metabolic profile of giant panda seminal plasma has not yet been
reported. The aim of the present study was to identify the whole
proteome profiles of giant panda seminal plasma using a gel-free,
label-free shotgun proteomics approach. Semen from four sexually mature
giant pandas (aged between 9 and 16 years, the average was 11.5\selectlanguage{ngerman}±3.32 )
was collected by electroejaculation during the breeding season according
to the previous methodology(Cai et al., 2018). Semen was collected into
a plastic container and immediately placed in a centrifuge. An aliquot
of 0.5 mL of fresh semen was centrifuged at 900 × \emph{g} for 30 min at
4\selectlanguage{english} to separate seminal plasma from spermatozoa. Seminal plasma was then
transported at 4\selectlanguage{english} and frozen at -80\selectlanguage{english} until further use. All samples were
collected during artificial insemination and cryogenically stored
following a standard, routine procedure at the Sichuan Key Laboratory of
Conservation Biology for Endangered Wildlife, Chengdu Research Base of
Giant Panda Breeding.

All samples were initially sonicated three times in ice and lysed in
lysis buffer containing 100 mM NH4HCO3(pH 8), 6 M Urea and 0.2\% SDS,
followed by 5 min of ultrasonication on ice. The lysate was centrifuged
at 12000 \emph{g} for 15 min at 4degC and the supernatant was transferred
to a clean tube. Extracts from each sample were reduced with 2mM DTT for
1 h at 56\selectlanguage{english} and subsequently alkylated with sufficient Iodoacetamide for
1 h at room temperature in the dark. Then 4 times the volume of
precooled acetone was mixed with samples by well vortexing and incubated
at -20degC for at least 2h. Samples were then centrifuged, and the
precipitation was collected. After washing twice with cold acetone, the
pellet was dissolved by a dissolution buffer containing 0.1 M
triethylammonium bicarbonate (TEAB, pH 8.5) and 6 M urea. Protein
concentration was determined again by Bradford protein assay.

The supernatant from each sample, containing precisely 0.12 mg of
protein was digested with Trypsin Gold (Promega) at 1:50
enzyme-to-substrate ratio. After 16 h of digestion at 37degC, peptides
were desalted with a C18 cartridge to remove the high urea, and desalted
peptides were dried by vacuum centrifugation.

Shotgun proteomics analyses were performed using an EASY-nLCTM 1200
UHPLC system (Thermo Fisher) coupled with an Orbitrap Q Exactive HF-X
mass spectrometer (Thermo Fisher) operating in the data-dependent
acquisition (DDA) mode. A sample volume containing 2 \selectlanguage{greek}μ\selectlanguage{english}g of total
peptides was injected onto a home-made C18 Nano-Trap column (2 cm\selectlanguage{ngerman}×100
\selectlanguage{greek}μ\selectlanguage{english}m, 3 \selectlanguage{greek}μ\selectlanguage{english}m). Peptides were separated on a home-made analytical column (15
cm\selectlanguage{ngerman}×150 \selectlanguage{greek}μ\selectlanguage{english}m, 1.9 \selectlanguage{greek}μ\selectlanguage{english}m), using a 60 min linear gradient from 5 to 100\%
eluent B (0.1\% FA in 80\% ACN) in eluent A (0.1\% FA in H2O) at a flow
rate of 600 nL/min. The detailed solvent gradient is listed as follows:
5-10\% B, 2 min; 10-30\% B, 49 min; 30-50\% B, 2 min; 50-90\% B, 2 min;
90-100\% B, 5 min. Q-Exactive HF-X mass spectrometer was operated in
positive polarity mode with a spray voltage of 2.3 kV and capillary
temperature of 320\selectlanguage{ngerman}°C. Full MS scans ranging from 350 to 1500 m/z were
acquired at a resolution of 60000 (at 200 m/z) with an automatic gain
control (AGC) target value of 3×10\textsuperscript{6} and a maximum ion
injection time of 20 ms. The 40 most abundant precursor ions from a full
MS scan were selected for fragmentation using higher energy collisional
dissociation (HCD) fragment analysis at a resolution of 15000 (at 200
m/z) with an AGC target value of 1×10\textsuperscript{5}, a maximum ion
injection time of 45 ms, a normalized collision energy of 28\%, an
intensity threshold of 2.2e4, and the dynamic exclusion parameter of 20
s.

The resulting spectra from each fraction were searched separately
against `P101SC18111984-01-ailuropoda\_melanoleuca.fasta' by the search
engines: Proteome Discoverer 2.2 (PD 2.2, thermo). The searched
parameters were as follows, a mass tolerance of 10 ppm for precursor ion
scans and a mass tolerance of 0.02 Da for the product ion scans were
used, carbamidomethyl was specified in PD 2.2 as fixed modifications,
oxidation of methionine (M) and acetylation of the N-terminus were
specified in PD 2.2 as variable modifications and a maximum of 2
miscleavage sites were allowed.

For protein identification, a protein with at least one unique peptide
was identified at FDR less than 1.0\% on peptide and protein levels,
respectively. Proteins containing similar peptides that could not be
distinguished based on MS/MS analysis were grouped separately as protein
groups. Precursor quantification based on intensity was used for
label-free quantification. Gene Ontology (GO) and InterPro (IPR)
analysis were conducted using the InterProScan-5 program against the
non-redundant protein database (including Pfam, PRINTS, ProDom, SMART,
ProSiteProfiles, PANTHER)(Jones et al., 2014), and the databases COG
(Clusters of Orthologous Groups) and KEGG (Kyoto Encyclopedia of Genes
and Genomes) were used to analyze the protein family and pathway. Based
on the related species, the probable interacting partners were predicted
using the STRING-db server (http://string.embl.de/). STRING is a
database of both known and predicted protein-protein
interactions(Franceschini et al., 2013). The enrichment pipeline (Huang
da et al., 2009) was used to perform GO, IPR and KEGG enrichment
analysis, respectively.

\textbf{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 discovered\emph{PAFAH1B1}plays 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.\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
Uniprot ID & Protein name & Gene symbol & Molecular
function\tabularnewline
\midrule
\endhead
G1LGH1 & WD repeat domain 1 & \emph{WDR1} & regulation of actin
cytoskeleton\tabularnewline
G1KZF6 & Receptor for activated C kinase 1 & \emph{RACK1} & regulation
of protein kinase C activity\tabularnewline
G1LLT4 & Platelet-activating factor acetylhydrolase IB subunit alpha &
\emph{PAFAH1B1} & regulation of microtubule cytoskeleton\tabularnewline
G1LGH6 & Coatomer subunit beta & \emph{COPB2} & a subunit of the Golgi
coatomer complex\tabularnewline
D2HI58 & Uncharacterized protein & unkown & unkown\tabularnewline
G1LI97 & Actin-related protein 2/3 complex subunit & \emph{ARPC1A} &
regulation of actin filament polymerization\tabularnewline
G1LI94 & unkown & unkown & unkown\tabularnewline
D2HX06 & Uncharacterized protein & unkown & unkown\tabularnewline
G1LEQ8 & SEC31 homolog A, COPII coat complex component & \emph{SEC31A} &
anterograde transport from the endoplasmic reticulum (ER) to the Golgi
apparatus\tabularnewline
D2HX51 & Coronin & unkown & unkown\tabularnewline
G1LEX2 & EMAP like 4 & \emph{EML4} & microtubule stabilization,
intracellular transport, and cell signaling\tabularnewline
G1M6T5 & Coatomer subunit alpha & \emph{COPA} & retrograde transport
from the Golgi to the endoplasmic reticulum (ER).\tabularnewline
\bottomrule
\end{longtable}

Overall, research on the seminal plasma proteome has the potential to
yield important insights into male reproductive biology and the
mechanisms underlying male infertility and other reproductive disorders.
As a vulnerable species, the reproduction of giant pandas has always
been a matter of great concern. Scientists have been studying ways to
increase the breeding rate of captive giant pandas to ensure their
population's health and sustainability. By establishing a dataset of the
giant panda seminal plasma proteome, and identifying specific proteins
and biomarkers associated with reproduction, researchers may be able to
develop new diagnostic and therapeutic approaches to improve male panda
reproductive health.

\textbf{Author Contributions:}

Conceived and designed the experiments: Kailai Cai, Yuliang Liu and Rou
Hou. Performed the experiments: Kailai Cai, Jiasong Chen, Feiping Li,
Shenfei Wang, Mengshi Zhang, Xianbiao Hu, He Huang, Junhui An, Donghui
Wang, Hairui Wang. Analyzed the data: Tao Wang. Contributed to writing
the manuscript: Tao Wang, Kailai Cai and Ayala James.

\textbf{Funding:} This study was conducted by the Chengdu Giant Panda
Breeding Research Foundation (CPF2017-17).

\textbf{Data Availability Statement:} The mass spectrometry proteomics
data have been deposited to the ProteomeXchange Consortium
(http://proteomecentral.proteomexchange.org) via the iProX partner
repository (Chen et al., 2022; Ma et al., 2019) with the dataset
identifier PXD044374.

\textbf{Conflicts of Interest:} The authors declare no conflict of
interest.

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\par\null

Figure 1. The Venn diagram displayed the annotation results of the
identified proteins in four databases (A), B and C showing the GO and
IPR annotations, respectively. The bar represents the number of
proteins.

Figure 2. The COG (A)and KEGG (B) annotation of detected seminal plasma
proteins. The bar represents the number of proteins.

Figure 3. The GO (A) and KEGG (B) annotation of panda seminal plasma
proteins and three other species (human, pig, sheep). (C) and (D)
represent the GO and KEGG annotation of panda-specific seminal plasma
proteins compared to three other species, The results of IPR analysis
have also been presented simultaneously (E).

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