Published online Jun 14, 2019.
https://doi.org/10.5534/wjmh.190012
Alterations of Spermatozoa Proteomic Profile in Men with Hodgkin's Disease Prior to Cancer Therapy
,1,2
Ashok Agarwal,1
Saradha Baskaran,1
Peter Natesan Pushparaj,3
Gulfam Ahmad,4
and Manesh Kumar Panner Selvam1
Abstract
Purpose
Hodgkin's disease (HD) is a type of cancer affecting men in the reproductive age with potential consequences on their fertility status. This study aims to analyze sperm parameters, alterations in proteomic profiles and validate selected protein biomarkers of spermatozoa in men with HD undergoing sperm banking before cancer therapy.
Materials and Methods
Semen analysis was carried out in healthy fertile donors (control, n=42), and patients diagnosed with HD (patients, n=38) before cancer therapy. We compared proteomic profiles of spermatozoa from donors (n=3) and patients (n=3) using LTQ-Orbitrap Elite hybrid MS system.
Results
A total of 1,169 proteins were identified by global proteomic in both groups. The ingenuity pathway analysis revealed that differentially expressed proteins involved in capacitation, acrosome reaction, binding of sperm to the zona pellucida, sperm motility, regulation of sperm DNA damage, and apoptosis were significantly downregulated in HD patients. Validation of proteins implicated in sperm fertility potential by Western Blot demonstrated that peroxiredoxin 2 (PRDX 2) was underexpressed (p=0.015), and transferrin (p=0.045) and SERPIN A5 (p=0.010) protein levels were overexpressed in spermatozoa of men with HD.
Conclusions
Findings of this study indicates that the key proteins involved in sperm fertility potential are significantly altered in men with HD, which provides substantial explanation for the observed low sperm quality in HD subjects prior to cancer therapy. Furthermore, our results suggest PRDX 2, transferrin and SERPIN A5 as possible candidate proteins for assessing sperm quality in HD patients prior to cancer therapy.
INTRODUCTION
Hodgkin's disease (HD) is one of the most prevalent cancers in men in the reproductive age. According to the American Society of Cancer [1], about 4,570 men are estimated to be diagnosed with HD and about 590 men are projected to die from HD. The five years survival rate in people diagnosed with this HD is about 86% [2] and hence, fertility preservation is crucial for men diagnosed with HD. Sperm cryopreservation is recommended to all male patients before cancer therapy [3, 4].
Lower sperm quality, such as sperm concentration or motility has been reported in HD patients even before cancer therapy when compared to healthy individuals [5, 6, 7]. Though the reasons for this lower sperm quality is not fully understood, psychological factors or immune-mediated mechanisms affecting the spermatogenesis have been hypothesized to be the possible contributing causes [8, 9]. Molecular changes at the subcellular level of spermatozoa that can explain the low sperm quality in men with HD are still unknown.
Proteomic analysis of spermatozoa is a high-throughput technique that elucidates the complex nature of the sperm [10, 11]. Advances in mass spectroscopy and analysis of proteomic profile using bioinformatic tools have been used in the discovery of potential biomarkers in tissues and biological fluids [10, 11, 12, 13, 14, 15]. Literature review reveals that the proteomic profile of spermatozoa in HD patients has not been investigated until now. Therefore, we sought to investigate the sperm proteomic profiling of HD subjects in comparison to healthy fertile volunteers in order to elucidate the reason(s) for low sperm quality in HD subjects at the time of sperm banking. The objectives of this study are: (1) to analyze and compare semen parameters of men with HD, who banked their specimen before cancer treatment, with fertile donors; (2) to characterize and compare the spermatozoa protein profile among cancer patients and healthy fertile donors; and (3) to validate proteins of interest implicated in fertility that are differently expressed in Hodgkin's cancer patients when compared to controls.
MATERIALS AND METHODS
1. Study subjects and ethics statement
This study was approved by the Institutional Review Board (IRB) of Cleveland Clinic. All the participants signed an informed written consent at the time of sample collection at the Andrology Center, Cleveland Clinic (No. #13-1554). Semen samples were obtained from 42 healthy male donors of proven fertility (control group) who had fathered a child in the past 2 years before enrollment in the study and 38 patients with HD before cancer therapy (cancer group). All the subjects included in the control group had normal semen parameters [16].
Men between the ages of 20 to 45 years diagnosed with HD were included in the study and these patients have not yet started their oncological treatment. We have excluded men with azoospermia and those under the supportive medication, steroids or drugs. In addition, patients with systemic reproductive tract inflammation, smoking, sexually transmitted disease, and leukocytospermic were excluded from the current study.
2. Semen analysis
Semen analysis was performed according to the World Health Organization (WHO) guidelines (2010) [16] in samples from both control (n=38) and cancer (n=33) groups. Semen samples were collected by masturbation after sexual abstinence of at least 48 hours. Samples were allowed to liquefy completely for 20 minutes at 37℃, and semen analysis was performed according to WHO guidelines [16] using the Microcell counting chamber (Vitrolife, San Diego, CA, USA) to evaluate sperm count, motility and round cells. For the samples from the control group, after completion of routine semen analysis, the remainder of the sample was centrifuged for 7 minutes at 1,000×g. The clear seminal plasma was aspirated from the samples and spermatozoa were stored at −80℃ for proteomic studies. Semen samples from cancer group were cryopreserved with Freezing Medium Test Yolk Buffer (TYB) using an established protocol [4].
3. Preparation of semen samples
Semen samples from the control group were thawed at room temperature and centrifuged at 3,000×g for 30 minutes to remove the remnants of seminal plasma and washed with phosphate buffer saline (PBS). Cryopreserved samples from the cancer group were thawed to room temperature and centrifuged at 3,000×g for 30 minutes to remove the TYB and the supernatant was discarded. The pellet was then washed with PBS and centrifuged at 3,000×g for 30 minutes. This step was repeated 3 times to remove the remains of TYB.
4. Protein extraction and quantification
Proteins were extracted with radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma-Aldrich, St. Louis, MO, USA) containing the proteinase inhibitor cocktail (Roche, Indianapolis, IN, USA). RIPA was added to the sperm pellet (approximately 100 µL for 100×106 sperm) and incubated overnight at 4℃ to allow the complete lysis of the spermatozoa. The supernatant was aspirated after centrifugation at 13,000×g for 20 minutes to a clean microcentrifuge tube. The protein concentration was determined using a commercially available kit, bicinchoninic acid kit (Thermo, Rockford, IL, USA), following the manufacturer instructions.
5. Preparation of semen samples for proteomics
Proteomic analysis was performed on randomly selected donor (n=3) and HD (n=3) samples. Further, normalization of the samples was done by pooling the samples to overcome the biological variation [17, 18]. The samples were mixed with sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) buffer and subjected to one-dimensional-PAGE in triplicate runs. After completion of electrophoresis, the gel was cut into 6 pieces and digested using 5 µL trypsin (10 ng/µL), 50 mM ammonium bicarbonate and incubated overnight at room temperature. The samples (cut pieces) were alkylated with iodoacetamine and reduced with dithiothreitol. The peptides from the digested gel were extracted in two aliquots of 30 µL acetonitrile (10%) with formic acid (5%). The two aliquots were pooled together and evaporated to <10 µL and then diluted with 1% acetic acid to make up a final volume to 30 µL.
6. Liquid chromatography-mass spectrometer analysis
Proteomic profiling of spermatozoa was carried out using a Finnigan LTQ linear ion trap mass spectrometer liquid chromatography (LC)-mass spectrometer analysis (MS)/MS system. The peptides were fractionated by injecting 5 µL volumes into high-performance liquid chromatography column (Phenomenex Jupiter C18 reversed-phase capillary chromatography column; Phenomenex, Torrence, CA, USA). Fractions containing the peptides were eluted in acetonitrile/0.1% formic acid at a flow rate of 0.25 µL/min and were introduced into the source of the mass spectrometer on-line. The micro electrospray ion source was operated at 2.5 kV. A full spectral scan was performed by utilizing the data dependent multitask ability of the instrument to determine peptide molecular weights and amino acid sequence of the peptides [19].
7. Database searching and protein identification
Tandem mass spectra generated by LC-MS/MS system were retrieved using Proteome Discoverer ver. 1.4.1.288. Mascot (ver. 2.3.02; Matrix Science, London, UK), Sequest (ver. 1.4.0.288; Thermo Fisher Scientific, San Jose, CA, USA) and X! Tandem (ver. CYCLONE 2010.12.01.1; The GPM, thegpm.org) search was performed on all the MS/MS raw files. The search was limited to the human reference sequences database (http://www.hprd.org/) assuming the digestion enzyme trypsin. The mass tolerance for parent ion was set to 10 parts per million (ppm) and for fragment ion with 1.0 Da. The search results were uploaded into the Scaffold (ver. 4.0.6.1; Proteome Software Inc., Portland, OR, USA) program as previously described [20]. Protein probabilities were assigned by the Protein Prophet (Systems Biology, Seattle, WA, USA) algorithm. Annotation of proteins was performed using Gene Ontology (GO) terms from National Center for Biotechnology Information.
8. Quantitative proteomics
The relative quantification of the proteins was performed by comparing the number of spectra, termed spectral counts in both cancer and control groups. To achieve false detection rate <1%, protein identification criteria was established at >99% probability as explained in our previous study [21]. The abundance of the proteins was determined by matching the spectra (spectral counts or SpCs), and classified as high (H), medium (M), low (L), or very low (VL). To overcome the sample-to-sample variation, normalization of spectral counts was done using the normalized spectral abundance factor (NSAF). In general, longer proteins have more peptide identifications than shorter proteins. NSAF ratio determines the actual expression of the protein in the samples. Proteins with ratio <1 and >1 are considered underexpressed and overexpressed, respectively. Different constraints for fold-change cutoffs and significance tests (p-value) based on the average SpC from 3 replicate runs were applied to obtain the differentially expressed proteins (DEPs) [20]. Appropriate filters were applied used to minimize the errors due to the presence of low abundance proteins. Abundance and the expression of DEPs are based on the following criteria: (i) VL - SpC range, 1.7–7; NSAF ratio (≥2.5 for upregulated and ≤0.4 for downregulated proteins); and p≤0.001, (ii) L - SpC range, 8–19; NSAF ratio (≥2.5 for upregulated and ≤0.4 for downregulated proteins); and p≤0.01, (iii) M - SpC range, 20–79; NSAF ratio (≥2.0 for upregulated and ≤0.5 for downregulated proteins); and p≤0.05, (iv) H - SpC, >80; NSAF ratio (≥1.5 for upregulated and ≤0.67 for downregulated proteins); and p≤0.05.
9. Bioinformatic analysis
DEPs identified in both the study groups were subjected to functional annotation and enrichment analysis using publicly available bioinformatic annotation tools and databases such as GO Term Finder, GO Term Mapper, UniProt, and Database for Annotation, Visualization and Integrated Discovery (DAVID) (http://david.niaid.nih.gov). Protein-protein interaction was demonstrated using Ingenuity Pathways Analysis (IPA) based on the criteria: experimental evidence, neighborhood, gene fusion, occurrence, co-expression, existing databases, and text mining. Proprietary software package Metacore™ (GeneGo Inc., St. Joseph, MI, USA) was also used to identify the upstream regulators involved in the enriched pathways. Based on their function and role in fertility potential in male six DEPs were chosen for validation by Western blot (WB).
10. Protein validation by Western blotting
The DEPs involved in the reproductive functions and fertilization process, and top canonical pathways were selected for validation. The function of these proteins are well described in the literature. From the identified proteins, six DEPs were selected for validation. The DEPs were validated by WB using n=6 for the control group and n=6 for cancer group. A total of 20 µg of spermatozoa protein was mixed with equal volume of loading buffer (125 mMol Tris-HCl, pH 6.8, 2% SDS, 5% glycerol, 0.003% bromophenol blue, and 1% β-mercaptoethanol). The sample mixture was boiled for 10 minutes and kept on the ice for 5 minutes. 30 µL of each sample was loaded into each well of a 4% to 15% SDS-PAGE and electrophoresed for 2 hours at 90 V along with a set of molecular weight marker (Sigma Chemical Co., St. Louis, MO, USA). The resolved protein bands were then transferred onto polyvinylidene difluoride (PVDF) membranes at 18 V for 30 minutes using a transfer buffer of 25 mMol Tris base, 192 mMol glycine, and 20% methanol. PVDF membranes were blocked with Tris-buffered saline-Tween-20 (TBST) with 5% bovine serum albumin and used for immunodetection of sperm proteins. For each protein analysis, specific primary antibodies were incubated at 4℃ overnight (Table 1). Next, the membranes were washed four times with TBST for 10 minutes and incubated with the secondary antibodies at room temperature for 1 hour (Table 1). Membranes were washed four times again with TBST (10 minutes) and finally treated with enhanced chemiluminescence (ECL) reagent (GE Healthcare, Marlborough, MA, USA) for 5 minutes. ECL reacted blots were exposed to Chemi-Doc (ChemiDoc™ MP Imaging System; Bio-Rad, Hercules, CA, USA) to detect the chemiluminescence signals.
Table 1
Primary and secondary antibodies used in this study
11. Total protein staining
The total amount of protein present in the membranes was identified using a Colloidal Gold Total Protein Stain (Bio-Rad). The protocol was performed according to manufacturer instructions. Briefly, the membranes were washed twice for 10 minutes in the distilled water and stained with total colloidal gold protein stain for 2 hours at room temperature by gentle shaking. Stained membranes were washed twice with distilled water for 10 minutes, and the densitometry image was captured using calorimetric mode on Chemi-Doc (ChemiDoc™ MP Imaging System; Bio-Rad).
12. Statistical analysis
Data analysis was performed using MedCalc Statistical Software (ver. 17.8; MedCalc Software, Ostend, Belgium). Mann–Whitney test was carried out to compare the semen parameters of control and cancer groups, and the results were considered significant with p<0.05. The same test was used to compare the expression levels of the proteins validated using WB technique in both the groups.
RESULTS
1. Semen parameters in men with Hodgkin's disease
Semen analysis revealed significant decrease (p<0.05) in sperm concentration, total count and total motile sperm count in cancer group when compared to control group (Table 2).
Table 2
Sperm parameter of men from control and cancer group
2. Proteomic shotgun analysis revealed a total of 1049 different peptides common to both experimental groups
In this study we had used LC-MS/MS, a high throughput technique to obtain the proteome profile of spermatozoa from men diagnosed with HD before cancer therapy and compared this profile with the proteome profile of fertile donors. LC-MS/MS spectrometric analysis identified a total of 1,169 different peptides, out of which a total 1,049 proteins were common to both experimental groups, 101 were unique to the spermatozoa of proven fertile donors, and 19 were unique to the spermatozoa of patients with HD before cancer therapy (Fig. 1A). Relative abundance of the identified proteins revealed that 508 in very low abundance, 280 in low abundance, 286 in medium and 95 in high abundance (Fig. 1B). Among the identified proteins, 134 were detected as DEPs between the two experimental groups (Table 3). The sperm proteome of men with HD before cancer therapy had 35 overexpressed and 80 underexpressed DEPs compared to the sperm proteome of fertile donors (Fig. 1B). In the proteomic analysis of our study, we were also able to identify 16 DEPs unique to fertile donors and 3 DEPs unique to men with HD (Fig. 1C).
Fig. 1
(A) Total number proteins identified in the spermatozoa of proven fertile donors' group and patients with Hodgkin's disease (HD) before cancer therapy by liquid chromatography-mass spectrometer analysis (MS)/MS spectrometry. (B) Distribution of the identified proteins based on their relative abundance. (C) Differentially expressed proteins (DEPs) of experimental groups.
Table 3
Differently expressed proteins identified in sperm proteome of men with HD compared with donors
3. Ingenuity pathways Analysis revealed cell-to-cell signaling and interaction as top biofunction dysregulated in spermatozoa of men with Hodgkin's disease
IPA analysis of the DEPs revealed that pathways associated with top diseases and biofunctions were dysregulated in HD group with a cut off equal to 4.44 and a p=0.01 (Fig. 2). The top three pathways identified in the diseases and biofunctions are cell-to-cell signaling and interaction, reproductive system development and function, and cellular assembly and organization (Fig. 2). Functional analysis of DEPs identified that majority of the sperm proteins were associated with sperm functions (Fig. 3). Binding of sperm and binding of zona pellucida processes were affected due to the aberrant expression of sperm proteins, such as some subunits of T-complex protein 1 (TCP1, CCT2, CCT3, CCT4, CCT6A, CCT7, and CCT8), acrosin (ACR), probable inactive serine protease 37 (PRSS37) and angiotensin-converting enzyme (ACE). Other DEPs have a connection between the binding to zona pellucida and binding to sperm, (Fig. 3A). Additionally, the DEPs (ACR, TCP1, CCT2, CCT3, CCT4, CCT6A, CCT7, and CCT8) were also involved in the carcinoma functions in spermatozoa of men with HD (Fig. 3B). IPA analysis also showed that the DEPs ACE and ACR were involved in several reproductive functions such as sperm binding, fertility and fertilization process of the spermatozoa (Fig. 3C). Furthermore, IPA analysis also showed that production of reactive oxygen species (ROS) with metabolism of ROS and synthesis of ROS were affected due to altered expression of several DEPs (IL4I1, ITGAM, ITGB2, LTF, MPO, PRDX2, PRTN3, RACK1, S100A8, SERPIN A1, TF, TRAP1, APOA4, FN1, and IDH1) (Fig. 3D).
Fig. 2
Diseases and biofunctions identified in the spermatozoa of proven fertile donors' group and patients with Hodgkin's disease before cancer therapy by Ingenuity Pathway Analysis with a cut off=4.44 and p=0.01.
Fig. 3
Ingenuity Pathway Analysis of the differently expressed protein in spermatozoa of proven fertile donors' group and patients with Hodgkin's disease before cancer therapy, with the connecting protein related to (A) binding of zona pellucida and binding of sperm; (B) biding of sperm and carcinoma; (C) binding of sperm, fertility and fertilization; and (D) metabolism, production and synthesis of reactive oxygen species. TCP1: T-complex protein 1 subunit alpha, CCT3: T-complex protein 1 subunit gamma, ACR: acrosin, PRSS37: probable inactive serine protease 37, ACE: angiotensin-converting enzyme, IL4I1: L-amino-acid oxidase, ITGAM: integrin alpha-M; IDH: isocitrate dehydrogenase, ITGB2: integrin subunit beta 2, LTF: lactotransferrin precursor, PRTN2: transcription regulatory protein 2, RACK: receptor for activated C kinase, S100A8: S100 calcium-binding protein A8, SERPIN: serine proteinase inhibitors, TF: transferrin, TRAP: heat shock protein 75 kDa mitochondrial percursor, APOA: apolipoprotein A, FN1: fibronectin, MPO: myeloperoxidase.
4. Western Blot validation of spermatozoa proteins in men with Hodgkin's disease
Based on selection criteria and the biological role, five proteins (ACE, PRDX2, CCT3, TF, and SERPINA5) were validated by WB. Our results showed that the expression of ACE was comparable in control and HD groups (Fig. 4A). The expression level of CCT3 was found to be similar in HD and control groups (Fig. 4C). Furthermore, our WB results revealed an underexpression (p=0.015) of PRDX 2 (Fig. 4D), while overexpression of TF (p=0.045) and SERPINA5 (p=0.010) in HD group when compared to fertile donor group.
Fig. 4
Protein expression levels of the differentially expressed proteins selected for validation by Western blot in spermatozoa of proven fertile donors' group and patients with Hodgkin Disease before cancer therapy. (A) Angiotensin converting enzyme (ACE); (B) T-complex protein 1 subunit gamma (CCT3); (C) Peroxiredoxin-2 (PRDX2); (D) Transferrin; (E) Plasma serine protease inhibitor (SERPIN A5). Results are expressed as mean±standard error of mean and in fold variation to donors' group.
DISCUSSION
Proteomic analysis using bioinformatic tools offers a comprehensive information regarding the protein distribution, and functional and molecular pathways associated with the peptides identified in spermatozoa [13]. Previous studies in the global proteomics analysis of spermatozoa resulted in the discovery of biomarkers for different pathologies [10, 13, 14, 15, 22]. In this study we used LC-MS/MS, a high throughput technique, to profile the spermatozoa proteins from men diagnosed with HD prior to cancer therapy and compared it with the proteome profile of fertile donors. The semen analysis was performed according to WHO guidelines (2010) [16], and the results indicated that the men from cancer group had lower sperm concentration, total sperm count and total motile count, while there was no significant difference in the motility. The quality of sperm in HD patients was lower when compared to fertile donors, however the sperm parameters values from patients were still within the WHO (2010) [16] reference values. The presence of lower sperm quality in men with cancer, particularly with HD obtained in our study is compliant with previous reports [5, 6, 7]. However, semen analysis alone can not predict whether the patients are fertile, subfertile or infertile. Semen parameters are used as a screening test to detect the contribution of male factor, but they fail to provide a full understanding of fertility potential [23, 24]. As several factors (hyperactivation, capacitation, acrosome reaction, oxidative stress, sperm DNA fragmentation, etc.) in addition to semen quality contribute to the ability of spermatozoa to fertilize an oocyte, use of high-throughput techniques can allow a better understanding of the spermatozoal fertility potential at molecular level. In the present study, proteomic profiling of spermatozoa showed altered expression of key proteins associated with sperm function, which may contribute to the diminished fertility potential seen in some HD patients.
To minimize the variability existent in the complex nature of the spermatozoa [12], we had pooled the samples and run it in triplicate. LC-MS/MS spectrometry identified a total of 1,169 different peptides in the two experimental groups and about 1,049 were common to both experimental groups. The proteomic profile of spermatozoa is not new and similar studies have been performed in mature and immature ejaculated spermatozoa from fertile men [14], infertile men [11], and in spermatozoa of infertile men with bilateral varicocele [22]. However, this is the first report on the sperm proteins in men with HD before cancer therapy. To find an explanation for the lower sperm quality in these patients, we had chosen five proteins for validation. These proteins were selected based on their involvement in reproductive functions and fertilization process. Cell to cell signaling and interactions are crucial for the fertilization process [25, 26], and the binding of sperm to zona pellucida is in fact crucial for the oocyte fertilization. Our analysis revealed that binding of zona pellucida and binding of sperm were dysregulated in spermatozoa of men with HD. ACE is a protein involved in fertility and our IPA analysis relates this protein to binding of sperm and zona pellucida, and interrelates the binding of sperm with fertilization process and fertility. ACE has been reported to be altered in infertile patients [13, 27, 28]. In our experiment, the proteomic analysis showed an overexpression in ACE, although the WB validation did not show any significant increase in expression. This protein has been described as a functional protein in sperm motility and capacitation [26, 29]. Due to the localization of ACE in the peri-acrosomal section of the sperm head, and an agonist-induced function in acrosomal exocytosis, this protein consequently has a significant role in the fertility process [30]. Studies have reported a correlation between decrease in ACE with an increase in ROS and possible impairment of acrosome reaction [28, 29]. PRDXs are central antioxidant enzymes and have a role in sperm function and male fertility [31]. PRDX2 is mostly found in the cytosol, eliminating ROS formed as a by-product of metabolism [32]. This protein was found to be underexpressed by the proteomic analysis and validated by WB. Since PRDX2 is an antioxidant enzyme, the underexpression of this protein can be related to low levels of oxidative stress. Transferrin which has been found to be overexpressed by proteomic analysis and validated by WB is also involved in sperm protection [33]. This protein decreases oxidative stress by reducing the availability of free iron [34].
The acrosome is crucial for the fertilization and it contains numerous hydrolytic enzymes, including ACR [35]. ACR is synthesized and stored in the sperm acrosome matrix in the form of proacrosin, an inactive form of ACR. During acrosome reaction, proacrosin transforms to intermediate and mature forms through autoactivation [36]. Previous studies have reported the importance of ACR in fertility and sperm ACR activity is significantly reduced in men with anti-sperm antibodies [37]. Another study reported that the ACR activity is a reflection of male fertility status [38]. ACR is a proteolytic enzyme capable of hydrolyzing the zona pellucida of oocyte, playing an important role in fertilization [39]. This is in line with our IPA analysis results which revealed that ACR was related to binding of sperm, zona pellucida and fertilization process. Our proteomics analysis demonstrated an under expression of ACR. The low expression of ACR in patients can be related with other protein identified by LC-MS/MS, the probable inactive serine protease 37 (PRSS37). PRSS37 is involved in acrosin activation [36], and abnormal activation of proacrosin/acrosin system has been reported in sperm with low levels PRSS37 [36]. Interestingly, our proteomic results showed an underexpression of PRSS37 (Table 3). SERPIN A5 was found to be overexpressed in spermatozoa of men with HD compared with proven fertile donors by LC-MS/MS spectroscopy with concordant results from validation. SERPIN A5 is localized in external plasma membrane and has many functions including inhibition of several serine proteases related to male reproductive tract [40, 41, 42]. This protein has a role in motility [40, 41], fertilization process [42], and indirectly inhibits the degradation of semenogelin1 and 2 [43]. Our proteomic study results demonstrated the overexpression of semenogelin1 and 2 (Table 3), which is concordant with the overexpression of SERPIN A5 that inhibits the degradation of these proteins. Besides these functions, SERPIN A5 also inhibits acrosin and indirectly protects the male genital tract from the damaging effects of excess acrosin [42].
CCT3 is a subunit of T-complex protein 1, and a chaperone responsible for protein folding in the cells that requires the assistance of enzymes [44]. Low levels of this protein are related to low cell proliferation in cell counts, and induced cell apoptosis [45, 46]. This fact can be indirectly responsible for the low sperm count in the HD group. Another fact to consider is the association between high levels of CCT3 and its negative correlation with survival in cancer patients [45, 46]. The expression levels of CCT3 are low in sperm of HD patients, which is in compliance with the high survival rate for HD described by the American Cancer Society, [1].
CONCLUSIONS
This study aimed to explain reasons for poor semen quality seen in men diagnosed with HD prior to cancer therapy, as understanding the cellular and molecular mechanisms of cancer-associated poor semen quality may guide future research in fertility preservation. Our proteomic data showed an altered proteomic profile in spermatozoa of men with HD and our WB results validated the vital proteins involved in the fertility process. Validated proteins can serve as potential biomarkers to determine the sperm quality and fertility status in HD patients prior to cancer therapy.
Conflict of Interest:The authors have nothing to disclose.
Author Contribution:
Conceptualization: AA.
Data curation: ADM, PNP
Formal analysis: ADM, MKPS, SB.
Investigation: AA.
Methodology: ADM, PNP.
Project administration: AA.
Resources: ADM, MKPS.
Software: PNP.
Supervision: AA.
Validation: ADM.
Writing—original draft: ADM.
Writing—review & editing: all authors.
ACKNOWLEDGEMENTS
Financial support for this study was provided by the American Center for Reproductive Medicine, Cleveland Clinic, OH, USA. Ana D. Martins was funded by FCT (SFRH/BD/108726/2015) and Fulbright (ID: E0585654). The authors would like to thank to: Dr. Belinda Willard (Director, Proteomics Core Laboratory, Lerner Research Institute, Cleveland Clinic) for her support in the proteomic analysis.
Data Sharing Statement
The data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study.
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ORCID IDs
Funding Information
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Fuel Cycle Technologies
SFRH/BD/108726/2015
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Fulbright Association
E0585654
