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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 5  |  Issue : 2  |  Page : 203-216

Qualitative and quantitative analysis of protein expression in the caput and cauda regions of the rat epididymis


1 Department of Reproductive Biology, AIIMS, New Delhi; Mergen BioLogics Training and Research Private Limited, Guwahati, Assam, India
2 Department of Biophysics, AIIMS, New Delhi, India; Department of Environmental Medicine and Public Health, Icahn School of Medicine at Mount Sinai, Manhattan, New York, USA
3 Department of Biophysics, AIIMS, New Delhi, India; Department of Neurology, Saint Louis University School of Medicine, Saint Louis, Missouri, USA
4 Department of Reproductive Biology, AIIMS, New Delhi, India
5 Department of Biophysics, AIIMS; Department of Biochemistry, School of Chemical and Life Sciences, Jamia Hamdard University New Delhi, India

Date of Submission22-Apr-2021
Date of Acceptance03-May-2021
Date of Web Publication16-Jun-2021

Correspondence Address:
Binita Basnet Baruah
Department of Reproductive Biology, AIIMS, New Delhi; Mergen BioLogics Training and Research Private Limited, Guwahati - 781 003, Assam
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_61_21

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  Abstract 


Background: The aim of this study was to identify the differentially expressed proteins in the sperm isolated from the caput and the cauda region of the rat epididymis. This is the first study on the quantitative nongel-based proteomics to have identified differentially expressed proteins in the cauda epididymal sperm. Material and Methods: This was achieved by isolation of sperm from the caput and the cauda of the rat epididymis followed by the tryptic digestion of the proteins and the resulted peptides were subjected to isobaric tags for relative and absolute quantitation-label and mass spectrometry (MS)/MS analysis. With the help of quantitative proteomics, we have been able to elucidate some of the major proteins involved in the process of sperm maturation. Results: In total, 999 proteins from the spermatozoa of caput and cauda region of the epididymis were identified. We have reported about 10 downregulated proteins and 15 upregulated proteins that have been in the sperm from the cauda region of the epididymis. HongrES1, the membrane of the SERPIN family specifically expressed in the principal cells of the cauda epididymis, have been found to be expressed three-fold higher. Conclusion: This study contributes to the understanding of the importance of different proteins at the different stages of the maturation during the transit of the spermatozoa. The higher and lower expression of different proteins in the epididymal region depicts their roles in priming the spermatozoa for normal fertilizing ability. Thus, the target proteins can be further studied for the possible development of male contraception.

Keywords: Caput, cauda, electrophoresis, epididymis, isobaric tags for relative and absolute quantitation, spermatozoa


How to cite this article:
Baruah BB, Kola S, Rukmangadachar L, Chaturvedi P, Alagiri S. Qualitative and quantitative analysis of protein expression in the caput and cauda regions of the rat epididymis. Biomed Biotechnol Res J 2021;5:203-16

How to cite this URL:
Baruah BB, Kola S, Rukmangadachar L, Chaturvedi P, Alagiri S. Qualitative and quantitative analysis of protein expression in the caput and cauda regions of the rat epididymis. Biomed Biotechnol Res J [serial online] 2021 [cited 2021 Jul 23];5:203-16. Available from: https://www.bmbtrj.org/text.asp?2021/5/2/203/318437




  Introduction Top


The ability to fertilize an oocyte can be attained by a sperm only when it has undergone a series of posttesticular modifications in the male as well as the female reproductive tract. During its transit through the epididymis, the sperm undergoes various biochemical and functional modifications, that facilitate its acquisition of motility and ability to be capacitated for fertilization.[1] Following the production of sperm in the seminiferous tubules, they are released into the lumen of the tubules where the flow of fluids transports them to the rete testis and subsequently to the efferent ducts. From there, the sperm pass through the different regions of the epididymis, i.e. the caput (head), corpus (body), and the cauda (tail), wherein they are stored until ejaculation. Thus, the epididymis serves three major functions: transport, storage, and the most important being the maturation of the sperm.[2],[3]

Each region of the epididymis has a distinct physiological function. Distinct structural features are displayed by the sperm present in each region. The sign of distinction between the sperm from caput and cauda is that caput epididymal sperm possess a cytoplasmic droplet at the proximal region of the flagellum that migrates toward the distal region and is eventually shed off during the transit.[4] Furthermore, there is remodeling of the acrosomal region in some species during the epididymal transit. It has been reported that the sperm from the cauda epididymis has the ability to move forward and bind to the zona pellucida after capacitation. However, caput epididymal sperm are not capable of fertilization.[5] The caput epididymal sperm are also characterized by weak, vibrational movements with no progression. The cauda epididymis acts as a sperm reservoir, whereas the caput and corpus are responsible for sperm maturation.[6],[7],[8]

During spermatogenesis gene transcription is an active feature however; the sperm entering the epididymis are transcriptionally and translationally silent and thus unable to generate new proteins.[9],[10] Thus, their functional maturation is highly dependent on their sequential interaction with specific components of the epididymal fluid in which they are bathed. Proteins are known to be acquired, deleted, or post-translationally modified as spermatozoa pass through the epididymis and this has been shown to be a critical factor in developing their ability to fertilize the oocyte.[11],[12],[13],[14],[15]

Proteomic analysis of mouse sperm proteome in the caput, corpus, and cauda epididymis has demonstrated that each of the proteins present in this fluid has a different distribution pattern along the organ.[16] A majority of the proteins, such as clusterin and transferrin, entering the epididymis from the rete testis disappear in the proximal caput epididymis[17] and has been reported to be replaced by secreted-clusterin and lactoferrin within the epididymis[18] suggesting that these components may be involved in protecting the spermatozoa against oxidative stress and/or bacterial attacks during their transit and storage. The presence of the main epididymal proteins in different mammals as reported are human epididymal protein (HE-1), epididymal-retinoic acid-binding protein precursor, epididymal secretory glutathione peroxidase precursor (epididymis-specific glutathione peroxidase-like protein), prostaglandin-H2 D-isomerase precursor, lipocalin protein (named LCN6).[17],[18] The role of these proteins in epididymal maturation is not well-characterized. They may act as signaling molecules for the regulation of epididymal activity as these proteins have been reported not to be binding with the spermatozoa. The objective of the present study was to analyze the proteins in the sperm from the caput and cauda regions of rat epididymis, with the help of both gel-based and nongel-based on proteomics tools.


  Materials and Methods Top


Cyanine dyes (Cy2, Cy3, and Cy5 were purchased from GE Healthcare, USA, DMF from spectrochem, Mumbai, India. CHAPS, urea, tris, acrylamide, bisacrylamide, TEMED, DTT, iodoacetamide and bromophenol blue were from Amresco, USA. Trypsin (mass grade) from Promega; acetonitrile and high-performance liquid chromatography grade water from G. Biosciences, USA.

Isolation of caput and cauda epididymal sperm

Institutional ethical approval was obtained for the use of Albino Wistar rats for this work. Adult rats (180–220 g) were sacrificed to remove the testes along with the epididymis. The epididymis was then separated and placed in 0.1M phosphate-buffered saline (PBS) (pH 7.4). The caput and cauda segments were removed and each of them was placed in a separate Petridish containing PBS. Each segment (caput and cauda) was teased separately and kept at 37°C for 30 min in the Petridish containing 0.1M PBS (pH 7.4) allowing the sperm to swim out in PBS. The sample containing the sperm was collected and centrifuged at 1500 g for 20 min. The pellet was resuspended in PBS containing protease inhibitor and was subjected to sonication.

Protein precipitation and quantification

The samples were mixed with four times volume of ice-cold acetone and kept at −20°C for 1 h protein precipitation. Protein pellets were air-dried and dissolved in lysis buffer (8M urea, 2M thiourea and 4% CHAPS). Protein concentration was determined by Bradford assay using BSA as standard.

SDS-PAGE

Proteins from sperms isolated from the caput and cauda of the rat epididymis were separated by SDS-PAGE using the Biorad electrophoresis system. SDS-sample buffer contained 62.5 mM Tris-HCl, 10% glycerol, 2% SDS, 5% β-mercaptoethanol, and 0.0125% bromophenol blue. The stacking and running gels contained 5% and 10% acrylamide, respectively. Electrophoresis was carried out at a constant current of 30 mA for 1 h. The gels were then stained with Coomassie blue R-250 and destained successively in the destaining solution (10% acetic acid, 40% methanol and 50% water). The protein profile of the caput and cauda epididymal spermatozoa from the left and right sides of the epididymis was also analyzed with the help of SDS-PAGE as described above and densitometry analysis by Image J software 1.47 h 23 versions (rsbweb.nih.gov/ij.html).

Two-dimensional gel electrophoresis

Before IEF, 200 μg of proteins in lysis buffer was added to rehydration stock solution (8M urea w/v, 2M thiourea w/v, 4% CHAPS w/v, 0.3% DTT w/v, 0.002% bromophenol blue v/v and 0.5% IPG buffer (pH 3–10 NL) v/v). After mixing, the samples were centrifuged at 7000 g for 2 min to remove any particulate matter. The solution was then loaded on a reswelling tray (Amersham Biosciences, USA). Immobiline Dry gel strip of pH range 3–10, 24 cm was used for IEF. Plastic cover on the strip was carefully removed and the gel surface was placed over the sample in the tray with forceps. The gel strip was overlaid with IEF cover fluid (Amersham Biosciences, USA) and was kept 16 h for optimum rehydration. The rehydrated IPG strips were kept in a strip holder and subjected for isoelectric focusing in an EttanIPGphor 3 IEF system (Amersham Biosciences, USA). IEF was stopped when a total volt-hours of 30,000 VhT was achieved. Temperature was set at 20°C. Strips were covered with mineral oil throughout the run period.

Before SDS-PAGE, each strip was equilibrated with SDS-equilibration buffer (6M urea, 75 mM tris, pH 8.8, 29.3% glycerol, 2% SDS, and 0.002% of bromophenol blue). Firstly with 10 ml SDS equilibration buffer containing 0.03% DTT for 15 min, followed by SDS equilibration buffer containing iodoacetamide for another 15 min. The strips were then loaded on 10% homogeneous acrylamide gel cast on SE 600 Ruby gel apparatus (Amersham Biosciences, USA). The strips were then sealed with agarose sealing solution to maintain continuity between the acrylamide gels and the immobiline dry strips. Separation was carried out with a constant current of 15 mA per gel at 20°C for 30 min, followed by 30 mA per gel at 20°C until the bromophenol blue dye front had runoff from the bottom of the gels.

Silver staining

After the two-dimensional gel electrophoresis, the gels were fixed overnight in 250 ml fixing solution containing 40% ethanol and 10% acetic acid. The gel was washed with 30% ethanol for 20 min followed by 15% ethanol and final washing with water; each step was performed for 20 min. This was followed by the addition of 250 ml sensitization solution containing 0.02% sodium thiosulphate for 1 min. The gel was rinsed three times with water for 20 s each with shaking. Subsequently 0.2% silver nitrate solution was added and the gel was gently agitated for 30 min. This step is light-sensitive, hence adequate protection was observed. The gel was rinsed three times with water for 20 s each. The developing solution (3% sodium carbonate, 0.05% formaldehyde and 0.005% sodium thiosulphate pentahydrate) was added and changed after 1 min with fresh solution to avoid background. This was kept until the development of spots, after which the reaction was terminated with stop solution (0.5% glycine). This was replaced with water after 20 min. The gel was washed three times with water for 10 min each.

Difference in-gel electrophoresis with cye dye labeling

Fifty micro gram of sperm protein from the caput and the cauda region of the epididymis were used for labeling with 200pmol of Cy3 or Cy5. Minimal labeling was performed by adjusting the pH of the protein solution to 8.8 with 50 mM Tris HCl and incubating the protein solution with 200 pmol of Cye dye on ice in dark for 30 min. Quenching of the unbound dye was carried out by treating with 1 μl of 10 mM lysine solution for 10 min on ice. Dye swapping was carried out so that Cy3 and Cy5 dyes labeling were distributed equally to prevent bias in protein labeling. Cy2 was used to label the same amount of internal standard (50 μg) resulting from the pooling of aliquots of all the sperm extracted from the caput and cauda region of the epididymis. The experimental design in this experiment is shown in [Figure 1]. Samples labelled with Cy dyes were mixed and reconstituted with rehydration buffer containing 8M urea w/v, 2M thiourea w/v, 4% CHAPS w/v, 0.3% DTT w/v and 0.002% bromophenol blue v/v and 0.5% IPG buffer (pH 3–10 NL) v/v. After mixing, the samples were centrifuged at 13,000 rpm for 2 min to remove the particulate matter if any. The solution was then loaded on a reswelling tray (Amersham Biosciences, USA). Immobiline Dry gel strip of pH range 3–10, 24 cm was used for IEF. The plastic cover on the strip was carefully removed and the gel surface was placed over the sample in the tray with forceps. The gel strip was overlaid with IEF cover fluid (Amersham Biosciences, USA) and were kept overnight for 16 h for optimum rehydration. Rehydrated IPG strips were kept on a strip holder and subjected to iso-electric focusing in an EttanIPGphor 3 IEF system (Amersham Biosciences, USA). IEF was stopped when a total volt-hours of 30,000 VhT was achieved. The temperature was set at 20°C. Strips were covered with mineral oil throughout the run period.
Figure 1: Experimental design of difference gel electrophoresis

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Image acquisition

Gels were scanned for Cy2, Cy3, and Cy5 using Typhoon TRIO (GE Healthcare, USA). Imaging of Cy2 was done at 488 nm excitation and 520BP40 emission filter; Cy3 images were scanned using 532 nm excitation and 580BP30 emission filter; Cy5 images using 633 nm excitation and 670BP30 emission filter. All gels were scanned with PMT setting of 700–800. Images were cropped using Image-Quant TM v5.5 to remove areas extraneous to the gel image.

DeCyder analysis

DeCyder software allows relative quantification of the expression. Expression differences within the gels (caput and cauda) are first calculated (differential in-gel analysis [DIA]) and then it is compared across the gels (biological variation analysis [BVA]) using the internal standard (Cy2) image as reference.

Differential in gel analysis

Gel images were processed using DeCyder version 6.5. The images were uploaded to the workspace using the image loader module. These images were imported to the DIA workspace to create four different workspaces or each gel pair for further processing. The maximum number of spots for each co-detection procedure was set to 1000. The spots on gels were co-detected automatically as 2D-DIGE image pairs, which intrinsically link a sample to its in-gel standard.

Biological variation analysis

The DIA workspaces were then imported to the BVA workspace for analyzing biological variation. The experimental setup and relationship between samples were assigned in DeCyder software in the BVA workspace. Each individual Cy3 or Cy5 gel image was assigned an experimental condition, either caput or cauda and all Cy2 images were classified as standards. The gel with the highest spot count was assigned as the master gel. Matching between gels was performed utilizing in-gel standard from each image pair. Matching was further improved by landmarking and manually confirming potential spots of interest.

Biological variation analysis normalization of spot volume and statistical analysis

The volume of a spot for a given dye is defined as the fluorescent intensity of the corresponding dye integrated over the area of a spot. Normalized volume refers to the volume normalized across the three dyes and across the gels. One of the outputs DeCyder provides is the ratio of the normalized volumes, also called the standardized abundances (Rpg = VolCy5 pg/VolCy2 pg, Gpg = VolCy3 pg/VolCy2 pg), for each spot P and gel g in the experiment. VolCy5 pg represents the normalized volume of spot p on gel g in the Cy5 sample and similarly for the other two dyes. The statistical analyses in DeCyder are based on the standardized protein log abundances, which are defined as the log10 of the standardized abundance.[19] In theory, the standardized log abundances follow a normal distribution and are comparable across all spots and gel using Student's t-test.

Analysis of protein abundance change between samples was performed using standardized log abundance value, the average ratio of the standardized log abundance was calculated for each spot. The mean of the standardized log abundance of spots on 2D gels in the caput and cauda gel sets were analyzed using Student's t-test.

Average ratio of spots

The average ratio for a given spot between caput and cauda region pools was generated in the DeCyder software. The degree of difference in standardized abundance between two protein spot groups is expressed as average ratio. The average ratio indicates the standardized volume ratio between the two groups. Values are displayed in the range of −α to −1 and +1 to +α. Values between-1 and +1 are not represented, hence a two-fold increase or decrease is represented by 2 and −2 respectively (not 2 and 0.5).

Protein identification

The spots of interest were picked manually from the corresponding silver-stained gels. The picked gel spots were destained with 30 mM potassium ferricyanide and 100 mM sodium thiosufate solution. Corresponding spots from the preparative gel were manually picked and destained with 50% acetonitrile and 25 mM ammonium bicarbonate solution. The destained spots were then treated sequentially with 100 μl of 100 mM ammonium bicarbonate and acetonitrile twice and finally, the dehydrated gel pieces were dried completely in speedvac to remove any residual acetonitrile. The gel pieces were then immersed indigestion buffer 25 mM ammonium bicarbonate containing 10 ng/μl trypsin and kept on ice. After 45 min of incubation, swollen gel pieces were covered with 50 μl digestion buffer and incubated overnight at 37°C. The next day the peptides were extracted from the gel pieces with 50% acetonitrile and 5% formic acid and lyophilized. The peptides then were reconstituted in 20 μl of 50% acetonitrile and 0.1% formic acid for analysis by ESI-Q-TOF-mass spectrometry (MS)/MS.

Mass spectrometric analysis

For online liquid chromatography (LC)-MS/MS analysis, the peptides were resuspended in 0.1% formic acid and 3% acetonitrile solution and injected to a Tempo™ nano-LC system (AB Sciex, Canada) equipped with a Michrom C18 75 μm × 150 mm column (MichromBioresources, Inc., USA). A 70 min gradient of increasing acetonitrile concentration was used to separate the peptides. The eluent was sprayed into a QSTAR XL tandem mass spectrometer (AB Sciex, Canada) and analyzed in information-dependent acquisition (IDA) mode using Analyst QS 1.1 software (AB Sciex, Canada).

Database searches

Mascot, version 3.2.2b, (Matrix Sciences, UK) was used for protein identification and quantitation from MS/MS data. For Mascot search, the nLC-ESI MS/MS data for 2+, 3+ and 4+ charged precursor ions were converted to centroid data, without smoothing, using the Mascot.dll script in Analyst QS1.1. The data were searched with a tolerance of 100 ppm for the precursor ions and 0.3 Da for the fragment ions. The following settings were used: trypsin was the cleavage enzyme, one missed cleavage allowed, carboxymethyl modification of cysteines was fixe modification and methionine oxidatio was elected as a variable modification. Spectra were searched against SwissProt 57.15 database (515203 sequences; 181334896 residues) with taxonomy: Rattus novergicus (rat).

Isobaric tags for relative and absolute quantitation labeling of the sperm proteins from the caput and the cauda region of the epididymis

A 100 μg of protein from sperm of caput and cauda was dissolved in 20 μl dissolution buffer TEAB (0.5M triethylammonium carbonate pH 8.5) and 1 μl of denaturant containing 2% SDS and vortexed. Further, 1 μl reducing reagent TCEP (tris-(2-carboxyethyl) phosphine) was added to each sample to make 5 mM TCEP final concentration. The tubes were incubated at 60°C for 1 h. Subsequently, the sample was centrifuged briefly and 1 μl freshly prepared 84 mM iodoacetamide solution was added and it was incubated in dark at room temperature for 30 min. A 1 mg/ml solution of trypsin was prepared and 10 μg was added to each sample. The samples were vortexed and spun followed by overnight incubation at 37°C. The isobaric tags for relative and absolute quantitation (iTRAQ) reagent was reconstituted and pH was adjusted to 7.8–8.5 with dissolution buffer. The content of each vial of iTRAQ reagent was transferred to each sample tube. The samples were mixed thoroughly by vortexing, centrifuged briefly and incubated at room temperature for 1 h. After 1 h incubation 100 μl of Milli-Q water was added to each tube to quench the iTRAQ reaction and was incubated at room temperature for 30 min. Contents from all iTRAQ labeled sample tubes were combined into one tube and mixed well by vortexing. The tube was dried and 100 μl water was added to the tube and reconstituted solution was mixed by vortexing. The sample was spun and dried completely. This step was repeated for a total of three times. Further 500–1000 μl cation exchange buffer containing 10 mM potassium phosphate in 25% acetonitrile at pH 2.5–3.0 was added and mixed. The pH was adjusted to 2.5-2.9 by the addition of orthophosphoric acid.

Strong cationic exchange high-performance liquid chromatography

1 ml of strong cationic exchange (SCX) buffer A (20% ACN/80% 10 mM potassium phosphate) was added and pH adjusted to 3 ml with orthophosphoric acid. Centrifugation was done at 15000 g at room temperature for 15 min. The supernatant was put into injection vial. The flow rate was adjusted to 400 μl/min. Initially, 100% methanol was run for 30 min, followed by water for 20 min, conditioning buffer for 60 min, and water for 15 min, 100% buffer B (buffer B - 20% ACN/80% 10 mM potassium phosphate, 500 mM potassium chloride, pH 2.9) pH adjusted to 3 by orthophosphoric acid) for 15 min. Equilibration was done by running 100% buffer A overnight at 50 μl/min or until ultraviolet chromatogram was stable. The standard peptides (beta gal, bovine serum albumin digest) were run to check column integrity and elution times. The sample was loaded and applied by auto-injection. Fractionation and elution of peptides were done as described by Mostovenko et al.[20] The SCX fractions were lyophilized. Each fraction was resuspended with buffer A since iTRAQ labeled peptides are bulkier, and more hydrophobic concentration of acetonitrile was increased to 5% in buffer A. The fractions were pooled into 8 fractions and desalted with a C18 spin column [Figure 2].
Figure 2: Gene ontology study of the biological and molecular functions of the proteins in the rat cauda epididymal spermatozoa when compared to caput epididymal sperm using Software Tool for Researching Annotations of Proteins software (http://cpctools.sourceforge.net)

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Mass spectrometric analysis

Digested peptides were solubilized in 50% acetonitrile containing 0.1% formic acid and loaded in a silica capillary (Proxeon Biosystem, USA) that fixed to QSTAR-XL-quadrupole/time-of-flight tandem mass spectrometer (Applied Biosystem, USA). The progress of each MS/MS run was monitored by recording the total ion current as a function of time for ions in the m/z range 140–1600. Mass spectra were acquired for 10 min setting the parameters by IDA method.

The source position and ion spray voltage were optimized. Further, the beta gal was used for optimization. 250 femtomol of beta gal on column should give at least 1000 mascot score with 100 ppm and 0.3 Da tolerance levels. In the MS/MS spectra ion range was adjusted in the range 100–1600 m/z. More collision energy is required to release iTRAQ reporter ions. This is set by going to script in the main menu of analyst and choose IDA CE parameters.dll. The intercept values were changed from default to +4 to account for higher collision energy to release reporter groups. It may be noted that very high values compromise score by fragmenting the high m/z ions, a low CE will not release reporter ions. MS/MS analysis and database search were performed using Protein Pilot 4.5 beta software using the Paragon database algorithm for search through mode. Searches were performed without constraining Mr or pI and allowed for the carbamido-methylation of cysteine, partial oxidation of methionine residues, and one missed trypsin cleavage. Search was further refined to include peptides with charges +2 and +5 and limited to Homo sapiens species. The peptide mass tolerance range was ± 1.2 kDa and fragment mass tolerance was ± 0.6 kDa. The confidence limit for the protein identification was set at a minimum of 95%.

Protein annotation

The identified proteins were annotated by submitting the accession numbers to the gene ontology (GO) tools. The accession numbers of all unique proteins identified in the dataset were analyzed using GO Slimmer tool from AmiGO Gene Ontology database (http://amigo.geneontology.org/cgi_bin/amigo/slimmer) and from Software Tool for Researching Annotations of Proteins (STRAP) an open-source protein annotation software for data visualization (http://cpctools. sourceforge. net). GO slimmer tool enable to map a protein list to a more restricted set of high level; broader parent GO terms, also known as GO slim terms. The results were derived into three separate categories-cellular component, molecular function, and biological process


  Results and Discussion Top


Protein profile of the sperm from the caput and cauda of the rat epididymis

The difference in protein profile between the sperm in the caput and cauda region of the epididymis was analyzed with the help of electrophoresis using 10% SDS-PAGE and stained with Coomassie R-250 [Figure 3]. Some of the differences in the protein profile were observed. For further confirmation, a 2D PAGE was performed. The difference in the protein profile of the sperm from the left and right caput and cauda region of the epididymis was also looked into. The gels were analyzed with the densitometry using the Image J software 1.47 h 23 versions (rsbweb.nih.gov/ij.html). However, no differences in the protein profile of the sperm from the right and the left epididymis were observed [Figure 4].
Figure 3: Ten percentage SDS-PAGE of Sperm taken from the different regions of the epididymis of rat (Lane 1: Protein Marker. Lane 2: Sperm sample from the Caput region of Epididymis of rat. Lane 3: Sperm sample from the cauda region of epididymis of rat)

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Figure 4: Densitometry study of the gel by Image J software1.47h 23 versions (rsbweb.nih.gov/ij.html)

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Differential expression of proteins by gel-based proteomics

The differential expression of proteins from sperm isolated from the caput region of the rat epididymis was compared with that of the sperm isolated from the cauda region of the rat epididymis [Figure 5]. For further confirmation, a two-dimensional difference gel electrophoresis (2D-DIGE) was performed. Sperm from caput and cauda were labeled with Cy3 and Cy5 respectively and the internal standard pooled from both caput and cauda was labeled with Cy2 dye. The experimental design of the 2D-DIGE of the gel is presented in [Figure 1]. A representative DIGE gel with three fluorescent images captured at three different excitation/emission wavelengths is shown in [Figure 6].
Figure 5: Two-dimensional electrophoresis of sperm from the caput and cauda region of the epididymis of rat

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Figure 6: Two-dimensional-difference gel electrophoresis of sperm from Caput and Cauda Region of Rat Epididymis (a. 50μg of Cy3-labelled caput sample. b. 50μg of Cy5 labelled cauda sample. c. 50μg of Cy2-labelled pooled standard) (equal amount proteins extracted from sperm of caput and cauda sample)

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Difference in-gel analysis

Intragel spot detection and quantification were performed using the DIA module of DeCyder 7.0 software. In DIA, the Cy2, Cy3 and Cy5 images for each gel were merged, spot boundaries were automatically detected and normalized spot volumes (protein abundances) were calculated. Any differences observed between gels due to electrophoretic artifacts were compensated for by normalization. During spot detection, the estimated number of spots was set at 1000 and nonprotein spots corresponding to the dust particles were manually removed. This analysis was used to calculate the abundance differences between the sperm of the caput and the cauda region of the rat epididymis [Figure 7]. The DIGE experiment resulted in the detection of 958 proteins [Figure 7] and [Figure 8]. Among them, 92 were increased and 18 were decreased. On careful examination of the statistical data by Student's t-test, however, only 17 protein spots were reported to be of significance. As the abundance of these proteins in the gel was deemed to be very low, we decided against going for further downstream analysis by MS for identification. To cover the proteome in depth and to get much better quantitative information, iTRAQ based quantitative proteomics experiments were planned [Figure 9].
Figure 7: Image of the differentially expressed spots of sperm proteins from rat caput and cauda. Marked with red are the protein spots down-regulated in expression, those with blue are up-regulated in expression and with green are the constitutively expressed proteins in both the samples

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Figure 8: Snapshot of differential in gel analysis workspace using DeCyder software. (a) Represents the image view of the gel (b) three-dimensional illustration of the selected spot (c) represents the spot statistics (d) protein table with the average ratio

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Figure 9: Strong cation exchange of isobaric tags for relative and absolute quantitation labeled proteins of the sperm from the caput and the cauda region of the rat epididymis by high performance liquid chromatography

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Identification of proteins in the sperm isolated from the caput and the cauda region of the epididymis

iTRAQ is a chemical labeling approach that incorporates stable isotopes into an NHS-ester derivative amine tagging reagent which when combined allows comparative, quantitative multiplexed analysis. The aim of this study was to identify the differentially expressed proteins in the sperm isolated from the caput and the cauda region of the rat epididymis. This was achieved by the following protocol: (1) isolation of sperm from the caput and the cauda of the rat epididymis, (2) the tryptic digestion of the proteins (3) the resulted peptides were subjected to iTRAQ-labeled (two-plex) with sperm from the caput labeled with 115 and sperm from the cauda were labeled with 117 (4) fractionation into 35 fractions through strong cation exchange which were pooled to 7 fractions by observing the peak range and the timing of elution and (5) MS/MS analysis.

The Paragon algorithm in ProteinPilot 4.5 beta software was used as the default search program with digestion enzyme set as trypsin and carbamidomethylation of cysteine residue, partial oxidation of methionine residues and one missed trypsin cleavage for the data analysis. The results obtained with at least 95% confidence from ProteinPilot software 4.5 beta were exported to Microsoft Excel for further analysis. 999 proteins from the spermatozoa of the caput and cauda region of the epididymis were identified. The proteins with 117/115 ratio ≤0.7 were considered to be downregulated in the sperm from the cauda region of the epididymis when compared to the sperm isolated from the caput region of the epididymis. The protein with a ratio greater than or equal to 1.3 were considered to be upregulated in the sperm from the cauda region of the epididymis when compared to the sperm isolated from the caput region of the epididymis. We have reported about 10 proteins that have been down-regulated in the sperm from the cauda region of the epididymis. We have reported about 15 proteins that have been upregulated in the sperm from the cauda region of the epididymis. This is the first study on the quantitative nongel-based proteomics to have identified differentially expressed proteins in the cauda epididymal sperm [Table 1] and [Table 2].
Table 1: List of upregulated proteins

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Table 2: List of downregulated proteins

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Differential expression of proteins in the cauda epididymal sperms

We have reported five proteins HongrES, beta-hexosaminidase, diazepam-binding inhibitor-like 5, epididymal secretory protein1, protein Zpbp [Table 3]. HongrES1, the membrane of the SERPIN family specifically expressed in the principal cells of the cauda epididymis, have been found to be expressed three-fold higher. On the transit of the sperm, it has been reported that this protein binds to the sperm head and thereby prevents the early capacitation.[21] In a knockout study of HongrES1 by Zhou et al.,[22] it has been demonstrated that knockout of HongrES1 results in premature capacitation leading to infertility and also in some cases deformed appearances of fetuses and pups. This indicates that HongrES1 plays a vital role in the elaborate regulation of sperm capacitation since precise regulation of capacitation. Two-fold differences in the expression of the proteins such as beta-hexosaminidase, diazepam-binding inhibitor-like 5, epididymal secretory protein1and protein Zpbp have been observed. These proteins play an important role in fertility and the presence of these proteins in the sperm of the cauda region indicates their involvement in the maturation of sperm and priming of the sperm for the fertilizing ability. Beta-hexosaminidase is an essential lysosomal enzyme of the epithelial cells of the rat epididymis. It catalyzes the hydrolysis of terminal sugar residues from a number of substrates such as glycoproteins, glycolipids, GM2 gangliosides, and glycosaminoglycans. This enzyme from the lumen of the epididymis gets bound to the sperm acrosome upon its journey through the caput to the cauda region. It serves to degrade substances endocytosed from the epididymal lumen and thereby modifying and creating a luminal environment wherein sperm can undergo their maturational modifications.[23],[24] In mouse, it has been reported that the sperm requires acrosomal-hexosaminidase to penetrate through the zona-pellucida by either of the two mentioned ways. This enzyme may remove terminal N-acetylglucosamine residues from the zona pellucida or it may function in a lectin-like manner to mask terminal N-acetylglucosamine residues, thereby blocking 131, 4-galactosyltransferase from binding zona glycoproteins.[25] Diazepam-binding inhibitor-like 5 protein has been reported to be present in the middle piece of the mature sperm tail enriched with mitochondria. This protein has been suggested to be involved in the regulation of several biological processes such as acyl-CoA metabolism; steroidogenesis, insulin secretion, and gamma-aminobutyric acid type A (GABA (A)/benzodiazepine receptor modulation indicating the role of this protein in providing the energy source for the spermatozoa.[26],[27] Epididymal secretory protein plays a major role in cholesterol metabolism by egressing the cholesterol from the endosomal or lysosomal compartment. Not much information is available about protein Zpbp. By the name, it can be inferred that this protein is involved in zona pellucida binding.
Table 3: List of proteins expressed two-fold higher out of 15 proteins to be up-regulated in the sperm from the cauda region of the epididymis

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The up-regulation of the proteins involved in energy metabolism such as glyceraldehyde-3-phosphate dehydrogenase, α-enolase, ATP synthase subunit alpha and O, mannosidase 2 alpha, saccharopine dehydrogenase, in the sperm very well correspond to the fact sperm isolated from the cauda region of the epididymis requires a large amount of energy in the form of ATP as it is in the cauda region that the sperm gains active forward motility and thus the presence of these enzymes play an important role in providing the energy fuel. It also indicates that the mitochondria of the cauda sperm are functionally more competent in comparison to the mitochondria of the immature sperm of the caput region. Three of the identified proteins enolase 1 α (ENO1), heat shock protein 70 (Hsp70), and ATP synthase subunit have also been identified by Baker et al.[28] Our results are also seen in the mouse by Ijiri et al.[29] They observed that ENO1 increases and Hsp 70 decreases during epididymal transit from caput to cauda in mouse as have been reported by our study.

Apart from the above proteins, certain proteins decreased in rat spermatozoa during epididymal maturation [Table 4]. Seven of the ten proteins are borderline cases. Three proteins are significantly reduced in expression level. The outer dense fibers (ODFs) are the sperm tail-specific cytoskeletal structures that provide tensile strength and protection to the sperm during the epididymal transit.[30],[31] Hence, it is quite surprising to observe the decrease in two of the isoforms of the ODFs in the cauda epididymal spermatozoa. Due to very little knowledge about the expression levels of ODPs and their relation with motility in rodent species the story remains unsaid. However, in human, Zhao et al.[32] have reported higher expression of ODFs isoform in case of asthenozoospermic men when compared to spermatozoa from normozoospermic men. This suggests that there is isoform-specific regulation during sperm maturation. Our observation could be like this however further study are required. The decrease in the Hsp 70 might signify the end of the translational activity and protein folding. This is expected as the sperm matures. Hence, with this study we could observe that journey of sperm is quite eventful in the epididymis with the gain and loss of proteins for the acquisition of sperm function.
Table 4: List of proteins significantly reduced in expression level

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Functional analysis of proteins identified in the caput and the cauda epididymal spermatozoa

With the help of quantitative nongel-based proteomics we have identified 999 proteins from the sperm of both the caput and cauda region of the epididymis. The proteins with a ratio of label 117/115, ≤0.7 were considered to be downregulated in the sperm from the cauda region of the epididymis when compared to the sperm isolated from the caput region of the epididymis. We have reported 10 proteins that have been downregulated in the sperm from the cauda region of the epididymis when compared to the sperm isolated from the caput region of the epididymis [Table 4]. We have reported about 15 proteins that have been observed to be up-regulated in the sperm from the cauda region of the epididymis when compared to the sperm isolated from the caput region of the epididymis [Table 3]. We could observe that in comparison to the total proteome only few proteins were differentially expressed. This indicates that sperm is trancriptionally and translationally inactive and the major changes that take place during the epididymal transit are due to the post-translational modification. This study is the first of its kind as no other study on the quantitative nongel-based differential expression of proteins in the sperm from the caput and cauda region of the rat epididymis has been carried out till date [Table 1] and [Table 2]. GO study of the 15 up-regulated proteins with the help STRAP software resulted in the categorization of proteins in the different biological functions [Figure 2].

In addition to these proteins, the identification of the metabolic enzymes in the cauda epididymal spermatozoa when compared to the caput epididymal sperm gives us an insight that the cauda epididymal sperm has an active mitochondrion. It is in the cauda region that the sperm gain active forward motility; hence, the upregulation of these metabolic enzymes is expected. The sperm maturation is accompanied by the activation of the mitochondria to provide ATP. The molecular function categorization of the up-regulated proteins indicated that most of the proteins in the binding activity, catalytic, enzyme-regulatory activity, and the antioxidant activity that scavenges the excess reactive oxygen metabolites released by spermatozoa during the epididymal transport [Figure 10]. The biological and molecular annotation provides information that the changes in the proteins of the sperm during epididymal maturation result in the acquisition of the ability of the sperm to bind to and fertilize the oocytes.
Figure 10: Network of interactions of proteins in rat epididymal sperm by ingenuity pathway analysis software

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As already stated, that epididymis has been dedicated to perform multiple functions such as sperm transport, elimination of defective sperms,[33] and finally sperm maturation and storage.[34] Thus, a functional epididymis is very much essential for fertility and therefore it will be more valuable to study the functions of these proteins identified to elucidate the aspects of male infertility and also to establish methods for male contraception. In a biological process, the identification of the molecules involved is the utmost requirement. It needs to be followed by the interaction and functions of the molecules. Thus, the functional analysis of proteins provides us with a better picture of the biological process under study. The involvement of different proteins during sperm maturation has been reported. Their functional analysis has provided us better information regarding the involvement of the proteins in the sperm maturation process in the epididymis. The epididymal maturation is the most important event in the production of healthy and fertile spermatozoa. The sperm undergoes various major modifications that are very much required for the acquisition of the fertilizing ability.[35],[36] Regardless, it is consequently maybe to be expected that the intraluminal proteome of the epididymis ranks among the most complex delivered by any endocrine gland.[37] The epididymis, particularly the initial segment, is absolutely essential for normal fertility. If this segment of the epididymis is missing, as in the case of the c-ROS knock-out mouse,[4] fertility is impaired. Thus, a functional epididymis is essential for fertility and for this reason, has been targeted by scientists trying to understand the etiology of spontaneous human infertility or develop novel approaches to male contraception.


  Conclusion Top


One of the important findings of this study is that epididymal sperm maturation is accompanied by the active role of mitochondria, suggesting that the presence of functional mitochondrial enzymes is important for the final maturation of these cells during capacitation. The list of proteins identified also accounts for the fact that the cauda epididymal sperms are motile and structurally mature when compared to the sperm from the caput region. This study contributes to the understanding of the importance of different proteins at the different stages of the maturation during the transit of the spermatozoa. The higher and lower expression of different proteins in the epididymal region depicts their roles in priming the spermatozoa for normal fertilizing ability. Thus, the target proteins can be further studied for the possible development of male contraception.

Financial support and sponsorship

Nil.

Conflicts of interest

The authors declare that none of the authors have any competing interests.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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