Abstract

Background

   During maturation, spermatozoa acquire motility and fertilizing
   capacity as they transit through the epididymis. In recent years,
   two-dimensional gel electrophoresis has been employed in proteomics
   studies conducted in rat, boar and human. However, there has not been a
   complete information regarding the proteins associated with sperm
   maturation in the epididymis. In this study, we employed iTRAQ
   proteomics to investigate proteins associated with sperm maturation
   between yak and cattleyak epididymis.

Results

   After a successful sampling and protein extraction, the iTRAQ coupled
   with LC-MS/MS mass spectrometry and bioinformatics analysis were
   performed. We identified 288 differentially abundant proteins (DAPs)
   between yak and cattleyak epididymis; 151 were up-regulated while 137
   were down-regulated in cattleyak relative to yak. Gene Ontology
   analysis identified that down-regulated DAPs in cattleyak were mostly
   enriched in the acetylation of protein component, along with negative
   and positive regulatory activities. iTRAQ proteomics data showed that
   the top up-regulated DAPs were mainly enriched in cell communication,
   cell adhesion, cytoskeleton organization, stress response,
   post-translational modifications and metabolic functions while the
   down-regulated DAPs were predominantly associated with sperm
   maturation, long-term sperm storage, sperm forward motility,
   sperm-oocyte fusion and regulatory functions.

Conclusion

   These results provide insight into the molecular mechanisms underlying
   male cattleyak sterility.

   Keywords: Yak, Cattleyak, Epididymis, Sperm maturation, iTRAQ
   proteomics

Background

   Cattleyak is the hybrid of cattle (Bos taurus) and yak (Bos grunniens)
   that demonstrates strong adaptability to harsh environmental conditions
   in the adjacent Alpine regions and the Qinghai-Tibetan Plateau in China
   [[45]1]. Cattleyak provides higher quantity of meat and milk as
   compared to cattle and yak. The male cattleyak is sterile and this may
   be associated with spermatogenic arrest [[46]2, [47]3] and/or some
   other factors such as genes or proteins expression dynamics along the
   epididymis. The sterility of male cattleyak phenomenon poses a major
   challenge and has restricted the hybridization procedure for decades.
   In previous years, not too many proteomics studies have been conducted
   to investigate male cattleyak sterility [[48]4, [49]5]. Most of the
   previous studies focused on testis and could not completely elucidate
   the exact cause of infertility in male cattleyak. Until now, no study
   has explored epididymis using iTRAQ proteomics as a means of
   identifying the sterility mechanism in male cattleyaks. Considering
   that testicular spermatozoa further undergo mandatory epididymal
   maturation in the epididymis, it is therefore a suitable organ for
   further investigating male cattleyak sterility.

   The epididymis is a long complex convoluted tube found on the surface
   of each testis, which connects the efferent duct to the vas deferens in
   the male mammalian reproductive tract [[50]6]. The epididymal
   epithelium consists of 5 main epithelial cell types: principal, clear,
   basal, narrow, and halo cells; which provide not only structural
   support to epididymis but also play significant role in the epididymal
   spermatozoa maturation process [[51]7]. The epididymis is the site
   where newly produced testicular spermatozoa acquire the ability to
   swim, penetrate and complete fertilizing competency. These functions
   are carried out from the secretion by epididymal epithelial cells
   (EECs) of several key proteins to create an interactive and dynamic
   microenvironment in the lumen where the spermatozoa are stored,
   protected, and undergo maturation. Moreover, Skerget et al. (2015)
   reported that, during sperm passage through the epididymis, the sperm
   surface protein must be acquired, lost, and modified to confer motility
   and fertilization competency to sperms [[52]8]. With their significant
   functions in the process of sperm maturation and male fertility, the
   proteins have received considerable attention and have been assumed to
   be prospective targets for identifying and treating infertility.
   However, until now, very limited knowledge is available about the exact
   roles of proteins in the regulation of the several important processes
   involved in the epididymal sperm maturation and fertilization.

   Advances in bioinformatics have greatly helped in understanding of
   sperm proteome composition and function. In recent years, some proteome
   studies have been successfully conducted in rat, human, and boar
   [[53]9–[54]11] by employing two-dimensional gel electrophoresis.
   However, the gel-based studies may suffer from drawbacks such as
   representation of the protein types at low concentrations and nature of
   the protein such as acidic or basic proteins [[55]5]. Isobaric tagging
   for relative and absolute protein quantification (iTRAQ) is a
   proteomics technique developed to quantitatively investigate protein
   abundance changes in different biological samples with high accuracy
   and reproducibility and the advantage of this multiplexing reagent is
   that 4 or 8 analysis samples can be quantified simultaneously [[56]12,
   [57]13]. In this study, we identified proteins associated with sperm
   maturation between yak and cattleyak epididymis employing iTRAQ
   proteomics.

Results

Identification of differentially abundant proteins (DAPs) between yak and
cattleyak epididymis by iTRAQ

   We applied a quantitative iTRAQ-based proteomics approach for the
   determination of proteomic changes between yak and cattleyak
   epididymis. For the identification and relative quantification of
   proteins, an overview of workflow described by yak and cattleyak
   epididymis proteome comparison is shown in (Fig. [58]1). In 3
   biological replicates, a total of 54,088 spectrums were generated and
   4596 proteins were identified from 23,801 peptides between yak and
   cattleyak epididymis proteome. Normally, the number of proteins
   decreased from 1452 to 80, as the increase in the protein coverage
   ranges from 0 to 100% (Fig. [59]2A). For the identification of peptide
   segments distribution, the Fig. [60]2B shows that most of the
   identified proteins contain < 25 peptide segments and the number of
   proteins segments decreased as the number of matching peptide segments
   was increased.

Fig. 1.

   Fig. 1
   [61]Open in a new tab

   The schedules of experimental treatment for the comparison of yak and
   cattleyak epididymal proteome. Proteins extracted from epididymis
   tissue samples were diminished, alkylated and enzymatically digested
   before labeling with iTRAQ, followed by LC-MS/MS mass spectrometry
   analyses. In the last section, proteins were identified and quantified
   before analysing and visualization. Legends: Male cattleyaks (n = 3;
   age: 1 year; named P1, P2, and P3) and Male yaks (n = 3; age: 1 year;
   named M1, M2, and M3)’’

Fig. 2.

   Fig. 2
   [62]Open in a new tab

   Peptide sequence coverage and identification of number of peptides. The
   A shows the proportion of proteins in different coverage ranges. The
   different colors representing different ranges of sequence coverage.
   The brackets show the number of proteins in different coverage ranges
   and their proportion to the total protein. The B shows the distribution
   of the number of peptides contained in the identified protein. The
   x-axis is the range of peptides covering the protein, and the y-axis is
   the number of proteins

Quantification of DAPs between yak and cattleyak epididymis by iTRAQ

   Out of the total identified proteins between yak and cattleyak
   epididymis proteome; 288 met the DAPs selection criteria, out of which
   151 were up-regulated (fold change ≥1.5, p ≤ 0.05) and 137 were
   p ≤ 0.05). Down regulated DAPs including Vitamin D-binding protein,
   DnaJ homolog subfamily C member 17, and Serotransferrin had fold
   changes of 0.06, 0.08 and 0.10 respectively. On the other hand, 34 out
   of the 151 up-regulated proteins had fold change values ≥2; among these
   were uncharacterized protein, A-kinase anchor protein 3,
   Uncharacterized protein, MHC class I antigen (Fragment), and Beta A4
   crystallin with fold changes of 4.6, 4.2, 3.9, 3.8, and 3.8
   respectively. We have listed top 30 up-regulated DAPs and top 30
   down-regulated DAPs identified from cattleyak relative to yak
   (Tables [63]1 and [64]2, respectively).

Table 1.

   The top 30 down-regulated differentially abundant proteins (DAPs)
   between yak and cattleyak epididymis
   Accession Description of Protein Protein symbol P value FC (P/M)
   F1N5M2 Vitamin D-binding protein GC 4.65E-09 0.062
   [65]Q2KI83 DnaJ homolog subfamily C member 17 DNAJC17 0.000100554 0.081
   G3X6N3 Serotransferrin TF 0.006778114 0.106
   [66]A0JNP2 Secretoglobin family 1D member (LppAB) SCGB1D 0.001735233
   0.181
   D4QBB3 Hemoglobin beta HBB 1.06E-05 0.225
   E1B9W6 Uncharacterized protein ADCY10 0.000214338 0.241
   [67]A7Z033 T-complex protein 11-like protein 2 TCP11L2 0.007235083
   0.256
   [68]P33433 Histidine-rich glycoprotein (Histidine-proline-rich
   glycoprotein) (HPRG) (Fragments) HRG 7.60E-07 0.272
   A5D7S6 PEF1 protein PEF1 0.001584413 0.286
   [69]Q3MHV8 RBM15B protein (Fragment) RBM15B 0.000115054 0.294
   B0JYP6 IGK protein IGK 0.002491825 0.295
   G3N1U4 Serpin A3–3 SERPINA3–3 4.24E-07 0.306
   [70]Q9XSK2 CD63 antigen (CD antigen CD63) CD63 7.98E-09 0.316
   [71]Q0IIG7 Ras-related protein Rab-5A RAB5A 0.000214338 0.318
   F6RF62 Uncharacterized protein MYL4 1.97E-08 0.344
   G9HQZ5 MHC class I antigen (Fragment) BoLA 0.000559995 0.362
   E1BND7 Uncharacterized protein PPFIA2 0.007269613 0.373
   [72]Q58D67 Dynactin 4 DCTN4 2.36E-05 0.403
   [73]Q3SZK1 Angio-associated migratory cell protein AAMP 0.004313819
   0.409
   E1BPI2 Non-specific serine/threonine protein kinase (EC 2.7.11.1)
   0.006717303 0.412
   [74]Q3ZCH5 Zinc-alpha-2-glycoprotein (Zn-alpha-2-GP)
   (Zn-alpha-2-glycoprotein) AZGP1 0.007264748 0.426
   A6QQR0 WDR75 protein WDR75 2.56E-05 0.427
   [75]Q2KJ61 Elongator complex protein 3 (EC 2.3.1.48) ELP3 0.000855102
   0.435
   E1BIM7 Uncharacterized protein CELF2 0.005137574 0.439
   E1BGN3 Histone H3 HIST2H3D 2.16E-15 0.444
   A1A4J3 Zinc finger, CCHC domain containing 3 ZCCHC3 0.000169658 0.449
   E1BE33 Uncharacterized protein ZEB2 1.58E-09 0.450
   G3MZD8 Uncharacterized protein 0.002250709 0.452
   E1BEP7 Uncharacterized protein ELP2 0.00728467 0.459
   [76]Q08E58 Tubulin tyrosine ligase-like family, member 12 TTLL12
   0.000147691 0.467
   [77]Open in a new tab

Table 2.

   The top 30 up- regulated differentially abundant proteins (DAPs)
   between yak and cattleyak epididymis
   Accession Description of Protein Protein symbol P value FC (P/M)
   E1BKY2 Uncharacterized protein 0.00881948 4.65
   F1MJS8 A-kinase anchor protein 3 AKAP3 0.000526461 4.26
   F1MIM1 Uncharacterized protein LOC104976250 0.000889658 3.89
   [78]Q3YJL1 MHC class I antigen (Fragment) BoLA 0.007469039 3.81
   [79]Q6DTZ8 Beta A4 crystallin (Beta-crystallin A4) CRYBA4 8.45E-09 3.80
   G3N2N9 Glutathione peroxidase GPX5 7.28E-05 3.55
   E1BE11 Uncharacterized protein HMCN1 5.28E-09 3.53
   F1MIM0 Uncharacterized protein 0.000618683 3.41
   [80]Q32KP8 Serine peptidase inhibitor-like, with Kunitz and WAP domains
   1 (Eppin) SPINLW1 1.30E-19 3.34
   [81]Q0VCG3 Parvalbumin alpha PVALB 0.000112643 3.03
   E1BGB7 Uncharacterized protein 1.07E-05 2.85
   [82]Q3MHJ9 Calcium/calmodulin-dependent protein kinase type II subunit
   beta (CaM kinase II subunit beta) (CaMK-II subunit beta) CAMK2B
   0.001572046 2.79
   [83]Q70IB2 Inactive ribonuclease-like protein 10 (Protein Train A)
   RNASE10 0.000205134 2.77
   F1MY32 Uncharacterized protein LY6G5C 0.004856315 2.76
   F1MSZ5 Uncharacterized protein ADAM28 1.08E-09 2.72
   [84]P02192 Myoglobin MB 3.62E-05 2.65
   [85]A6H742 Plastin-1 PLS1 7.90E-07 2.64
   G3N2L2 Uncharacterized protein RCN1 0.000161915 2.58
   F1MTV5 Amino acid transporter SLC1A5 0.004519988 2.56
   F1N5W4 Uncharacterized protein ENPP5 0.000276382 2.52
   F1N2E1 Uncharacterized protein WFDC8 0.0076419 2.45
   [86]Q2TBR5 Protein FAM166B FAM166B 2.10E-08 2.42
   [87]Q0VCU3 Cathepsin F CTSF 0.002189062 2.33
   A7E340 Mucin 15, cell surface associated MUC15 7.89E-33 2.32
   E1BD73 Poly [ADP-ribose] polymerase (PARP) (EC 2.4.2.30) PARP4 5.16E-14
   2.31
   F1MG20 17-beta-hydroxysteroid dehydrogenase type 6 HSD17B6 0.002590687
   2.26
   F1MV86 G1/S-specific cyclin-D3 CCND3 0.000102853 2.25
   A6QQ08 SNCA protein (Fragment) SNCA 0.009021835 2.24
   F1MUC1 Uncharacterized protein ABCC4 4.25E-06 2.21
   E1BJ49 Uncharacterized protein MASP2 0.000448626 2.18
   [88]Open in a new tab

Gene ontology (GO) analysis of the differentially abundant proteins

   Gene ontology (GO) analysis was conducted to better understand the
   functions of the all down-regulated and up-regulated DAPs identified by
   iTRAQ between yak and cattleyak based on biological process, cellular
   component, and molecular function categories. 35 GO terms were
   significantly enriched for down-regulated DAPs, including 22 biological
   process terms, 6 cellular component terms, and 7 molecular function
   terms. Majority of these down-regulated enriched GO terms relating to
   biological processes were mainly associated with acetylation of protein
   components, negative regulatory activity, and positive regulatory
   activity, from which top three listed enriched GO terms were negative
   regulation of endopeptidase activity (p = 0.002923), negative
   regulation of peptidase activity (p = 0.003519) and histone H3
   acetylation (p = 0.005172). According to the down-regulated cellular
   components, most of enriched GO terms were associated with
   acetyltransferase complex, among which the top three listed enriched GO
   terms include histone acetyltransferase complex (p = 0.002818),
   acetyltransferase complex (p = 0.004352) and protein acetyltransferase
   complex (p = 0.004352). From down-regulated molecular functions,
   majority of enriched GO terms were involved in inhibitor activity and
   regulator activity, among which top three listed enriched GO terms
   include peptidase regulator activity (p = 0.011867), antigen binding
   (p = 0.020786), and endopeptidase inhibitor activity (p = 0.021568)
   (Fig. [89]3A), while, among the up-regulated DAPs, 48 GO terms were
   significantly enriched between yak and cattleyak epididymis, including
   18 biological process terms, 7 cellular component terms, and 23
   molecular function terms. Most of the up-regulated DAPs enriched GO
   terms in particular to biological processes were involved in metabolic
   processes, protein localization and catabolic processes, among which
   top three listed enriched GO terms include organonitrogen compound
   catabolic process (p = 0.004521), receptor-mediated endocytosis
   (p = 0.005261) and cellular modified amino acid metabolic process
   (p = 0.006143). With respect to cellular component, the most important
   up-regulated enriched GO terms were lysosome and sperm part, in which
   top three listed enriched GO terms were lysosome (p = 0.001189), lytic
   vacuole (p = 0.001189) and blood microparticle (p = 0.005987).
   Significantly up-regulated enriched GO terms based on molecular
   function were associated with binding, inhibitor activity, and
   hydrolase activity, in which top three enriched GO terms were hydrolase
   activity (p = 1.26E-05), vitamin D binding (p = 0.000388) and steroid
   binding (p = 0.001698) (Fig. [90]3B).

Fig. 3.

   [91]Fig. 3
   [92]Open in a new tab

   GO classification of DAPs in cattleyak compared to yak. The A describes
   GO terms of down-regulated DAPs and the B describes the GO terms of
   up-regulated DAPs. The x-axis represents the number of GO terms and
   y-axis represents p-value of each GO terms

KEGG pathway enrichment analysis of DAPs

   Pathway analysis for identified proteins can deepen our understanding
   of the metabolic capacity of the species, biological processes
   information, and related diseases. In this study, we have mapped 87
   down-regulated DAPs and 136 up-regulated DAPs to the reference pathways
   in the KEGG database to determine biological pathways related to sperm
   function. In total, five up-regulated DAPs were significantly enriched
   in KEGG pathways whereas none of the down-regulated DAPs were
   significantly enriched in KEGG pathways. For the up-regulated DAPs, the
   enriched KEGG pathways were associated with Glutathione metabolism
   (p = 9.75E-05), Lysosome (p = 0.000102), Glycan degradation
   (p = 0.000646), Glycosaminoglycan degradation (p = 0.01678) and Thyroid
   hormone synthesis (p = 0.021941) (Fig. [93]4).

Fig. 4.

   Fig. 4
   [94]Open in a new tab

   KEGG pathway enrichments analysis of DAPs in cattleyak with respect to
   yak. The x-axis displays each of the enriched pathways. The y-axis
   shows the number of DAPs in each pathway

Protein-protein interaction (PPI) of DAPs

   In this study, the Search Tool for the Retrieval of Interacting Genes
   /Proteins 11.0 (STRING 11.0) database was used for identifying
   protein-protein interaction network of DAPs. After removing unconnected
   and self-loops proteins, the resulting PPI network generated 64 protein
   nodes and 90 edges (in Fig. [95]5). Furthermore, the network data file
   was directly imported into Cytoscape software for visual editing with a
   threshold value of a combined score > 0.7. DAPs that connected higher
   than others in created network were considered as hub proteins
   (Fig. [96]6); these hub proteins may have significant roles in the
   regulation of the network. In the PPI network, the top four hub
   proteins include vesicle-associated membrane protein 8 (VAMP8),
   SERPINA3–3, GGH, and DNAJC3.

Fig. 5.

   [97]Fig. 5
   [98]Open in a new tab

   Protein-protein interaction (PPI) network of DAPs in cattleyak created
   by STRING database

Fig. 6.

   [99]Fig. 6
   [100]Open in a new tab

   Protein-protein interaction (PPI) network of DAPs created with STRING
   database and visualized with Cytoscape. Proteins in blue were
   down-regulated and those in yellow were up-regulated. The line that
   connected the proteins with each other is called an edge. The color of
   the edges indicates the combined interaction score (edge score). The
   nodes with green borders described hub proteins

Elisa

   ELISA was performed to verify the results obtained from iTRAQ
   proteomics. The up-regulation of GGH, RAP1, GPX5 and MUC15 in cattleyak
   with respect to yak determined by iTRAQ was confirmed by ELISA
   (p < 0.05). Meanwhile, ELISA also verified the decreased expression of
   CD63, ELP3, LSM5 and GSTM1 in cattleyak compared to yak as identified
   by iTRAQ (p < 0.05) (Fig. [101]7). All these data validated the results
   obtained from iTRAQ.

Fig. 7.

   [102]Fig. 7
   [103]Open in a new tab

   Validation of iTRAQ data with ELISA. The vertical axis indicates the
   fold change of protein abundance. The horizontal axis indicates the
   methods for ELISA and iTRAQ. In each method, the protein expression
   level of yak was the control and designated as 1 to determine the
   expression level of cattleyak. Expression level of each protein is the
   mean of three replicates for cattleyak and plotted (mean ± SD) in
   relation to yak as control (Y = 1). The expression pattern of the
   protein CD63, ELP3, LSM5, GSTM1, GGH, ERAP1, GPX5 and MUC15 was
   validated respectively. *Protein expression were significantly
   different from control (p < 0.05, Student’s t-test)

Discussion

   Proteomics technologies present major possibilities to investigate the
   molecular mechanisms that regulate the functional activities of the
   sperm [[104]14]. Initially, the iTRAQ proteomics technique was used to
   study male cattleyak infertility with an increased focus on the testis.
   Until now, no research has used the aforementioned methodology to
   analyze the epididymis, which is recognized to be morphologically and
   functionally active in sperm maturation and fertility. In this study,
   iTRAQ proteomics is employed in the analysis of proteins associated
   with sperm maturation in the epididymis of yak and cattleyak. The
   epididymis serves important functions, including post-testicular sperm
   maturation which is important as it provides suitable environment for
   spermatozoa to develop progressive movement capacity and fertilization
   competence. The dynamic nature of the epididymal environment ensures
   that problems in the signaling pathways lead to infertility. In the
   epididymis the sperm protein, lipid and small RNA content are highly
   modified as a result of the continuous interaction with luminal
   proteins mainly secreted by the epididymal epithelium and extracellular
   vesicles, epididymosome [[105]15]. Epididymal maturation happens as
   structural modifications in the epididymis occur, allowing sperm to
   capacitate in the female reproductive tract [[106]16]. This study
   focuses on epididymal proteins and shows how they affect sperm
   functions. However, there exist major group of proteins identified to
   be expressed in testis as well as epididymis and they play specific
   important roles in sperm functions.

Epididymal sperm maturation

   The functional analysis identified a number of down-regulated DAPs
   involved in epididymal maturation in cattleyak (Table [107]1).
   Phosphatidylethanolamine-binding protein 4 (PEBP4) was studied in
   fertile, low fertile, and infertile bulls and found that the novel
   seminal PEBP4 expression was significantly higher in fertile bulls
   relative to low fertile and infertile bulls, implying that PEBP4 could
   play important roles in spermiogenesis, epididymal sperm maturation,
   and sperm motility [[108]17]. The existing evidence suggests that,
   down-regulation of PEBP4 in cattleyak could compromise sperm maturation
   processes and result in infertility. CD63, is a recognized exosome
   marker that is found in the apices of epididymal epithelial principal
   cells and the epididymal microenvironment, allowing for long-term sperm
   storage [[109]18]. The protein facilitates signal transduction
   functions, which are important in regulating cellular proliferation,
   growth, and motility. Downregulation of CD63 in cattleyak could have a
   significant impact on sperm storage and lead to fertility problems.
   Seminal plasma contains proteins associated with sperm progressive
   motility, including Zinc-alpha-2-glycoprotein (ZAG) and could play
   functional roles during maturation of spermatozoa, from the epididymis
   through fertilization in the female reproductive tract [[110]19]. The
   downregulation of ZAG in cattleyak suggests that sperms may well not
   develop progressive motility after ejaculation through into female
   reproductive tract.

   Glutathione S-transferase Mu 1 (GSTM1–1) and Fetuin-B were also found
   to be down-regulated in cattleyak compared to yak. The possible role of
   Glutathione S-transferase Mu 1 (GSTM1–1) in the epididymis is
   unspecified, despite the fact that it is a member of the Glutathione
   S-transferase mu class (GSTM1) found on sperms. GSTM1 is known to be
   associated with sperm-oocyte fusion and promotes precise joining of
   sperm surface-active components with the zona pellucida (ZP) during the
   sperm-egg fusion process [[111]20, [112]21]. Decreased GSTM1–1
   expression could be a potential cause of reduced or impaired
   fertilization in cattleyak. Fetuin-B was identified in mice as a plasma
   protein inhibiting ovastacin, a cortical granula protease known to
   cause ZP hardening and infertility [[113]22, [114]23]. It is possible
   that downregulation of Fetuin-B in cattleyak resulted in similar
   effects. The biological roles of the aforementioned proteins indicate
   that their downregulation could have a detrimental impact on sperm
   maturation, long-term sperm storage, sperm forward motility, and
   sperm-oocyte interaction.

Epididymal dysfunction

   In general, the epididymis comprises a variety of molecules and
   proteins that aid sperms in their quest to fertilize oocytes. Nuclear
   pores, which are essential in the exchange of macromolecules such as
   proteins and RNAs, facilitate interaction between the nucleus and the
   cytoplasm in eukaryotic cells. The nuclear localization signal (NLS),
   which is made up of 100 different proteins, particularly Importin
   subunit alpha, mediates active nuclear protein transport by recognizing
   karyophile proteins and forming a stable complex known as the nuclear
   pore-targeting complex (PTAC) [[115]24, [116]25]. Importin subunit
   alpha’s biological activity indicates that increased expression of this
   protein may block the critical contact between nucleus and cytoplasm in
   cattleyak epididymal cells, causing infertility. Mucins form highly
   glycosylated proteins which are classified into two types: secretory
   mucins and membrane-bound mucins. Mucin 15 have been discovered to be a
   membrane-bound mucin expressed by epithelial cells, implying that
   changes in membrane-bound mucins could affect cellular development,
   differentiation, transformation, invasion, and adhesion [[117]26].
   Mucin 15 upregulation in cattleyak suggests that Mucin 15 could perhaps
   enhance adhesion, which may affect or interfere with sperm cell
   migration through the epididymis. GAS2 was discovered to be a part of
   the microfilament system, associated with cell growth arrest through a
   phosphorylation mechanism during the Go-G1 transition [[118]27]. GAS2
   overexpression causes membrane ruffling and the termination of mitosis
   [[119]28]. The upregulation of GAS2 in cattleyak could have affected
   the vital molecules or cells participating in the overall growth of
   sperms in the cattleyak epididymis.

   Aminopeptidase and Poly (ADP-ribose) polymerase (PARP) were also found
   to be up-regulated in cattleyak and are known to be involved in a
   variety of stress responses. Aminopeptidase, an enzyme encoded by the
   Endoplasmic reticulum aminopeptidase 1 (ERAP1) gene, was thus
   discovered to play a key role in immune response [[120]29].
   Aminopeptidase perform functions similar to acute-phase proteins thus,
   it is secreted into the blood in response to inflammatory stimuli and
   is involved in increasing nitric oxide (NO) synthesis as a host defense
   mechanism [[121]30]. Poly (ADP-ribose) polymerase (PARP) functions as a
   DNA repair enzyme present in a variety of tissues, including the
   epididymis and it is activated when a DNA strand splits as a result of
   reactive oxygen species (ROS) or oxidative stress [[122]31, [123]32].
   PARP is activated by nicotinamide-adenosine dinucleotide (NAD) which
   results in ADP-ribosylation of PARP. Excessive PARP activation has been
   attributed to NAD+ depletion and corresponding cellular ATP depletion,
   eventually leading to necrotic-type cell death [[124]33]. This could be
   explained that increased Aminopeptidase and PARP expression levels led
   to an increase in a constant stress state of sperm cells involving the
   acquisition of certain toxic substances formed by altered DAPs.
   Moreover, mammalian epididymal spermatozoa achieve fertility
   development by a mechanism known as post-translational modifications
   [[125]34]. Polypeptide N-acetylgalactosaminyltransferase 6 (GalNAc-T6)
   is a member of the polypeptide N-acetylgalactosaminyltransferase
   (GalNAc-Ts) family that take active part in catalyzing the transport of
   N-acetyl—D-galactosamine: polypeptide (GalNAc) from the sugar donor
   uridine diphosphate (UDP)-GalNAc to the serine and threonine residues
   of glycoproteins [[126]35]. Previous research has shown that the
   modulation of spermatozoa plasma glycoproteins is essential for the
   development of functionally mature sperms during migration through the
   epididymis [[127]36, [128]37]. As a result, up-regulation of GalNAc-T6
   could modulate glycosylation and have a significant negative effect on
   sperm motility and perhaps on the fertility process.

   Proteolytic cleavages of surface proteins by protease enzymes are
   essential for the interactions of spermatozoa and zona pellucida.
   Protease inhibitors regulating the sperm membranes prevent premature
   capacitation, thereby controlling the proteolytic cascades [[129]38].
   Serpin A3–8 was discovered in buffalos as a protease inhibitor and
   served active in the protease inhibition mechanism [[130]39]. An
   essential Serine peptidase inhibitor-like protein (M/P = 3.339198) with
   Kunitz and WAP domains 1 (Eppin) were discovered in male monkeys and
   the high-titer monkeys were found to be infertile [[131]40]. As a
   result of the increased expression of these protease inhibitors, they
   could inhibit proteolytic cleavages needed for the early events of
   fertilization. Consequently, the biological functions of the proteins
   were associated with cell communication, cell adhesion, cytoskeleton
   organization, stresses, and post-translational modifications.

Regulatory mechanisms

   Different luminal environments exist within the epididymis, an
   undifferentiated epithelium undergo various sequence of modifications
   to become completely differentiated, with differences in anatomy, gene
   regulating activities, and functions [[132]41]. The mechanisms involved
   in gene expression are regulated at many different levels, from
   transcription to protein post-translational modifications. These highly
   complex processes necessitate the involvement of a variety of key
   proteins that are responsible for the activation of critical pathways.
   GO biological function analyses revealed a number of down-regulated
   DAPs involved in regulation and acetylation. The central objective of
   control in gene expression mechanisms is the transcriptional level,
   where histones and their acetylation in nucleosomes play functional
   roles in regulating gene expression. Histone H2A and its variant Core
   histone macro-H2A were identified as members of histone octamer that
   formed a structural unit (nucleosome) of chromatin [[133]42, [134]43].
   Histone H2A possesses N-terminal tail (H2ANtT) and C-terminal tail
   (H2ACtT). The H2ANtT perform essential functions mainly in
   inter-nucleosome interaction [[135]44], while H2ACtT recruits linker
   histone H1 to the nucleosome and stabilizes it [[136]45]. Deletion of
   H2ACtT could increase H2A mobility and also decrease nucleosome
   stability. Elongator complex protein 3 (ELP3) was found as a catalytic
   subunit of Elongator protein encoding a histone acetyltransferase and
   facilitating histone acetylation [[137]46]. Acetylation of histones
   permits chromatin to reveal DNA binding sites and initiate
   transcription [[138]47]. As a result, it is possible that the
   down-regulation of Histone H2A and ELP3 in cattleyak is a source of
   transcriptionally silenced genes that encode essential epididymal sperm
   maturational proteins.

   Post-transcriptional regulation of genes could be controlled by the
   processes of deadenylation and pre-mRNA splicing. CCR4-NOT
   transcription complex subunit 7 or CCR4-associated factor 1 (CAF-1)
   serves as a deadenylase enzyme in the mRNA degradation process, during
   which poly (A) tail is eliminated and the newly synthesized mRNA is
   highly decreased from 200 to 250 nucleotides to 10–60 nucleotides
   [[139]48, [140]49]. The poly (A) tail length is considered important in
   mRNA stability, translation efficiency, and gene regulation [[141]50],
   whereas U6 snRNA-associated Sm-like protein LSm5 serves as a major
   component of spliceosome initiating significant functions in pre-mRNA
   splicing mechanisms, resulting in the development of maturated mRNA
   [[142]51]. Multiple protein isoforms could be derived from a single
   gene during the pre-mRNA splicing process, and they could play an
   important role in cellular differentiation and organism growth
   [[143]52]. Down-regulation of U6 snRNA-associated Sm-like protein LSm5
   could facilitate the disruption of normal cellular functions, resulting
   in infertility. Furthermore, Aspartyl-tRNA synthetase (AspRS) and
   Protein transport protein Sec61 subunit gamma were discovered to be
   active in translational and post-translational processes, respectively.
   Aminoacylation is the mechanism by which tRNAs are charged prior to
   initiating the translation process. AspRS is an aminoacyl-tRNA
   synthetase which charges its cognate tRNA with aspartate amino acid and
   is involved in the oxidative stress response [[144]53]. However,
   transport protein Sec61 subunit gamma which is a component of
   protein-conducting channel (PCC) resides in the membrane of the
   endoplasmic reticulum, where it binds to translating ribosomes for
   co-translational protein transport [[145]54]. The biological functions
   of the translational and post-translational proteins revealed that
   their down-regulation may result in the inability of proteins to fold
   efficiently in the endoplasmic reticulum (ER).

Metabolic functions

   Metabolism involves series of chemical and physical modifications that
   develop in the body of animals, providing energy for the synthesis of
   newly required life processes. GO biological function analysis revealed
   a number of up-regulated DAPs involved in certain metabolism processes
   in cattleyak. It was previously reported that targeting sperm and
   energy metabolism have a stronger relationship [[146]55]. We recognized
   specific significant candidates for up-regulated DAPs involved in
   metabolic functions in this study: Biliverdin reductase A (BVR A) and
   Gamma-glutamyl hydrolase (GGH). In mammalian epididymis, Biliverdin
   (BV) and its reduced form, bilirubin (BR), perform essential functions.
   BVRA was identified as an enzyme that converted biliverdin to bilirubin
   and therefore considered to play a crucial role in scavenging reactive
   oxygen species (ROS), reactive nitrogen species (RNS), and nitric oxide
   through its substrate (BV), which is also a by-product (BR) [[147]56,
   [148]57]. The increased expression of BVRA in the epididymis of
   cattleyaks could restrict the capacity and quantity of biliverdin to
   facilitate certain oxidant activities. Proteins, micro and
   macronutrients all play important roles in fertilization; folate is a
   micronutrient that remains essential for sperm quality. Sperm
   aneuploidy has a negative impact on sperm production and could be
   reduced by consuming 400 mg to 700 mg total folate per day [[149]58].
   Gamma-glutamyl hydrolase (GGH) was discovered to be a lysosomal enzyme
   active in dietary folylpoly-y-glutamate metabolism, allowing
   folylmonoglutamate to reach the intestinal membrane to play a
   significant functions in folate cellular homeostasis [[150]59]. The
   biological function of GGH suggests that overexpression of this protein
   could compromise cellular folate maintenance and have a negative impact
   on cattleyak sperm production.

Conclusion

   We identified a total of 4596 proteins between yak and cattleyak
   epididymis. Out of the total, 288 were DAPs, of which 151 were
   up-regulated DAPs and 137 were down-regulated DAPs. Most of the
   identified proteins in cattleyak were involved in epididymal sperm
   maturation, epididymal dysfunction, regulatory and metabolic functions.
   iTRAQ proteomics data identified several key DAPs (PEBP4, CD63, ZAG,
   GSTM1–1, and Fetuin-B) that potentially play significant roles in the
   sperm maturation process in cattleyak. These results provide insight
   into the molecular mechanisms underlying male cattleyak sterility as
   upregulation and downregulation of certain important DAPs in the
   epididymis impede the normal development of a fertile ejaculate and
   represent a part of the causes of infertility. Continuously
   understanding the specific functional descriptions of the key DAPs may
   facilitate proper breeding techniques. Numerous DAPs from this study
   may be useful for further studies on the molecular mechanisms causing
   male cattleyak sterility.

Methods

Animals and epididymis sample collection

   Samples were collected under license in compliance with the Chinese
   Guidelines for the Care and Use of Laboratory Animals, and all
   procedures were authorized by the Southwest University of Science and
   Technology’s Institutional Review Board [[151]60]. Male yaks (Maiwa
   yaks) (n = 3; age: 1 year; named M1, M2, and M3) and Male cattleyaks
   (Maiwa yak × Tibetan taurine) (n = 3; age: 1 year; named P1, P2, and
   P3) were sampled from Maiwa yak population fed on a farm in Hongyuan
   county, Sichuan province of China.

   Veterinary surgery was used to extract epididymides from yaks and
   cattleyaks, fat and connective tissues were removed. Epididymides were
   separated apart from testis by fine-scale dissection and preserved in
   liquid nitrogen (− 196 °C), transported to the laboratory and stored at
   − 80 °C until further analysis. After the samples were collected, the
   animals were treated and stored in a safe and enclosed area near to the
   farm-house.

Total protein extraction from epididymal tissue

   Total protein was extracted from the epididymal tissue that contained
   sperm. Cryogenic grinding with mortar and pestle of each epididymal
   tissue sample was performed and for protein stability, lysis buffer
   (7 M urea, 2 M thiourea, 0.1% CHAPS) was added and vortexed to mix.
   Ultrasonic extraction of lysates, centrifugation and storage followed
   [[152]61]. The spectroscopic Bradford protein assay was used to measure
   the concentration of the extracted protein. Sample dilution in lysis
   buffer was performed for the final concentration within the range of
   the standard curve. BSA was dissolved with lysis buffer to a series of
   standard protein concentrations. Separate diluted sample 10 μL
   (standard) reacted respectively with 300 μL protein quantitation dye
   for 15 ~ 20 min. Simultaneously, absorbance was measured and sample
   concentrations were calculated based on the standard curve [[153]5].

Enzymatic digestion of the proteins

   100 μg of total protein sample was used by a filter-aided sample
   preparation (FASP) protocol. Addition of 10 μL of reducing reagent to
   the samples at 37 °C for 1 h and 2 μL of cysteine-blocking reagent for
   30 min at room temperature followed. The reductive alkylated protein
   solution was then added to a 10 K ultrafiltration tube (Merck
   Millipore, REF UFC501096), centrifuged at 12,000 rpm for 20 min, and
   bottom solution of the collection tube was discarded. Afterwards,
   100 μL dissolution buffer was added in the iTRAQ kit and centrifuged at
   12,000 rpm for 20 min. Prior to collection tube replacement, the
   settled solution in the collection tube was discarded, and there were
   three repetitions. Subsequently, 4 μg (1:50 compared to protein)
   trypsin (Promega REF V5111) in a volume of 100 μL was added and waited
   to react [[154]5]. Sample centrifugation at 12,000 rpm for 15 min was
   carried out on the following day. Subsequent to enzymatic digestion,
   the peptide solution was retained at the bottom of the collection tube.
   Ultrafiltration tube was used with 200 μL of ddH2O and centrifugation
   at 12,000 rpm for 15 min followed. Resulting 500 μL of the digested
   sample was collected from the collection tube.

iTRAQ labeling

   iTRAQ reagent (8-plex) was set to reach room temperature. 150 μL of
   isopropanol was added to each tube of iTRAQ reagent and vortexed to mix
   before centrifuged. A new centrifuge with 50 μL of sample (100 μg of
   enzymatic product) was transferred, iTRAQ reagent was added, vortexed
   and centrifuged. pH testing followed with 0.5 μL of the solution on pH
   paper of pH range 7.0 to 10.0. The tubes were then incubated at room
   temperature for 2 h. To stop the reaction, 100 μL of ddH2O was added to
   the mixture.

Offline pre-separation of enzymatically digested peptides and LC-MS/MS mass
spectrometry

   After dissolving the labelled lyophilized peptides [[155]5], the
   samples were centrifuged at 14,000 rpm for 10 min, and the supernatant
   was collected for use. Assessing the isolation conditions followed and
   100 μL of the total prepared sample was loaded at a flow rate of
   0.7 mL/mins and separation gradient were used [[156]61]. Protein
   analysis and the nanoscale reversed-phase chromatography was
   facilitated by mass spectrometer (Thermo, Model: Fusion). The high pH
   reversed-phase resulting segments were reconstituted with 20 μL (2%
   methanol, Sigma-Aldrich, article number: 14262, USA), (0.1% formic
   acid, Sigma-Aldrich, Cat. No. 56302, USA), and 13,000 rpm
   centrifugation for 10 min followed. Loading by sandwich technique,
   isolation flow along with isolation gradients followed [[157]60]. The
   mass spectroscopy (MS) parameter was set as: spray voltage 2.1 kV;
   capillary temperature: 250 °C, and Scan range: 350-1800 m/z.

Data analysis

   Bovine reference genome (ARS-UCD1.2) was used, and Fusion mass
   spectrometry was used for mass spectrometry analysis of iTRAQ. The
   original mass spectrometry data were processed uisng Thermo’s
   commercial software Proteome Discoverer 1.4. Search parameters were
   used and Mascot facilitated an automated decoy database search by
   selecting the decoy checkbox [[158]5]. The dataset collected proteins
   that existed in all three biological replicates. The observed findings
   were based on 95% percent confidence intervals for protein recognition
   as calculated by a Mascot probability study. Proteins with confidence
   intervals greater than “identity” were considered to be known. At least
   one distinct peptide aided in protein recognition. Protein
   quantification was determined by proteins with at least two distinct
   spectra.

   For protein quantification, it was required that a protein contains at
   least two unique spectra. MASCOT was used for Quantification protein
   ratios. The significant differentially abundant proteins (DAPs) were
   screened by T-test and proteins with a p-value less than 0.05 and a
   fold difference greater than 1.5 (Up regulated) or less than 0.67 (Down
   regulated) were considered DAPs.

GO, KEGG and PPI analysis of DAPs

   Gene Ontology (GO) terms in the database
   ([159]http://www.geneontology.org/) were used for mapping of the DAPs
   between yak and cattleyak epididymal tissues. Bonferroni Correction was
   used to normally adjust the p-value. Enrichment of biological pathways
   of the DAPs utilised KEGG database (Kyoto Encyclopedia of Genes and
   Genomes database). String Protein Interaction Database was used and the
   differential protein interaction network data files were complemented
   with Cytoscape software for visual editing.

Enzyme linked immunosorbent assay (ELISA)

   ELISA confirmed eight differential abundance proteins, namely CD63,
   ELP3, LSM5, GSTM1, GGH, ERAP1, GPX5 and MUC15. The total protein of
   each sample was extracted according to the manufacture’s protocol of
   DNA/RNA/protein co-extraction Kit (Tiangen Biotech (Beijing) Co., Ltd.,
   China). The different sample protein concentration was detected by
   NanoDrop 3000 Spectrophotometer (Thermo Fisher Scientific, Wilmington,
   DE, USA) and adjusted to 0.1 mg/mL. ELISA detection followed [[160]5]
   and standard curve as well as regression equation were made using the
   manufacturer’s standard samples with each sample analyzed with
   triplicates. Each well’s OD value detection was noted using microplate
   reader, set to 450 nm within 15 min. Using the regression equation, the
   targeted protein concentrations were calculated and each protein
   content was analyzed from each sample. Statistical analyses were
   performed using SPSS 22.0 standard version (IBM, Armonk, NY, USA).
   Student’s t-test was employed to analyze differences of each protein
   expression level between the control (yak) and tested groups
   (cattleyak). For all tests, statistical significance was taken as
   p < 0.05.

Acknowledgements