e n t e r of o r R epr d uc ti v e a n e Germ Cell Tumor ... · 1American Center for Reproductive...
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1 American Center for Reproductive Medicine, Urology, Cleveland Clinic, Cleveland, OH, 2 Center of Excellence in Genomic Medicine Research, Jeddah, Saudi Arabia, 3 College of Medicine, Prince Sattam Bin Abdulaziz University, Riyadh, Saudi Arabia, 4 Physiology, University of Health Sciences, Lahore, Pakistan 5 Department of Urology, Cleveland Clinic, Cleveland, OH Ashok Agarwal, PhD. 1 , Peter N. Pushparaj, PhD. 2 , Gulfam Ahmad, PhD. 1,3,4 , Muhammad K. Abu-Elmagd, MD. 2 , Mourad Assidi, PhD. 2 , Edmund S. Sabanegh., MD 5 , and Rakesh Sharma, PhD. 1 Identification of Sperm Proteins Associated With Infertility in Men With Seminoma of Germ Cell Tumor Using LTQ-Orbitrap Elite Hybrid Mass Spectrometry System ABSTRACT Objectives: Testicular germ cell tumor Seminoma (SGCT)) is the most common type of testicular cancer affecting young adult men. In these men, the semen quality begins to decline along with disturbances in the reproductive hormones. There is a lack of literature examining proteomic changes at the spermatogenic level in samples banked prior to cancer treatment. We set out to identify the sperm proteins and their role in altered semen quality in SGCT patients by proteomics and bioinformatics tool. Study Design: Identify key sperm proteins in patients with SGCT. Materials and Methods: Cryopreserved semen samples from men with SGCT (n=5) and normal healthy donors (n=5) before cancer treatment were preprocessed for proteomic analysis by LC-tandem Finnigan LTQ-Orbitrap Elite hybrid MS system. CID spectra were explored against the human reference sequence database. The relative quantity of these proteins was estimated by spectral counts (SC). The in silico examination was done using the Ingenuity Pathway Analysis (IPA) software (Qiagen, Redwood City, CA, USA) to deduce DEPs specifically linked with reproductive function. The statistical significance was calculated using Fisher’s Exact Test (P<0.05) with Benjamini-Hochberg (B-H) Multiple Testing Correction and the negative log P-value cut-off was set at 2. Results: 393 differentially expressed proteins were identified in the SGCT group. The sperm proteins associated with acrosome reaction (ACR, NSF, and SLC25A1), spermatogenesis (CCT6B, RUVBL1, SPA17, SPAG6, and SPANXD), binding of gonadal cells (ANXA5, CALR, ITGAM, ITGB2, LTF), spermiogenesis (DPY19L2, SPAG6), sperm adhesion molecule (SPAM1) involved in hyaluronic acid induced cell signaling and zona pellucida binding, sperm motility (AKAP4, ATP1A4, ROPN1B), were downregulated in SGCT group. The top differentially regulated protein network in TCS showed the downregulation of chaperons containing TCP1 subunits (CCT2, CCT3, CCT4, CCT5, CCT6A, CCT7, CCT8) that are vital for ATP-dependent folding of various proteins, including actin and tubulin in the spermatozoa. Conclusions: The proteomic analysis of semen samples in SGCT patients before cancer treatment indicates the downregulation of essential proteins involved in spermatogenesis, spermiogenesis, acrosome reaction, sperm-oocyte binding and sperm motility function. This partly explains the poor sperm quality observed in these patients even prior to the initiation of cancer therapy. Studies have shown that more than 90 percent testicular cancers originate from germ cells. Testicular germ cell tumor Seminoma (SGCT)) is the most common type of testicular cancer affecting young adult men. SGCT is a heterogeneous type of cancer with varied histopathological and clinical manifestations. The etiology of SGCT is largely unknown. In men with SGCT, the semen quality begins to decline along with disturbances in the reproductive hormones. There is a lack of literature examining proteomic changes at the spermatogenic level in samples banked prior to cancer treatment. Here, we set out to identify the sperm proteins and their role in altered semen quality in SGCT patients by proteomics and bioinformatics tools. INTRODUCTION After getting institutional approval, cryopreserved semen samples from men with SGCT (n=5) and normal healthy donors (n=5) were obtained from the Andrology Biorepository for the proteomic analysis using Finnigan LTQ-Orbitrap Elite Hybrid Mass Spectrometry. The samples were mixed with SDS Page buffer and separated on a 1D gel. Protein were digested in-gel using trypsin and peptides were extracted and evaporated to <10 μL. 30 μL was used for LC-MS analysis. Peptides eluted from the column were introduced into the source of the mass spectrometer on-line. The micro electrospray ion source is operated at 2.5 kV. The digest was analyzed using the data dependent multitask capability of the instrument. The data was analyzed, by using the CID spectra to search the human reference sequence databases with the search program Mascot and Sequest. These search results were then uploaded into the program Scaffold. The relative quantity of these proteins in the spermatozoa of SGCT patients was determined by comparing spectral counts (SC) of proteins in healthy controls. The DEPs were then analyzed using Ingenuity Pathway Analysis (IPA) software (Qiagen, USA) to decipher the differentially regulated canonical pathways, upstream regulators, causal networks, novel networks, diseases and biofunctions. The statistical significance was calculated using Benjamini Hochberg Multiple Testing Correction (P<0.01) and the negative log P-value cut-off was fixed at 2. Besides, the comparison analyses were performed using DEPs, upregulated and down regulated proteins to further understand the proteins implicated in reduced fertility in SGCT. MATERIALS and METHODS 1. We identified 393 proteins that were differentially regulated in the spermatozoa of men with SGCT compared to NC. 2. Significantly downregulated spermatozoal proteins were implicated in the canonical pathways (P<0.01); cellular assembly and organization, cell-to-cell signaling and interaction and reproductive development and function and mitochondrial dysfunction. Proteins involved in production of nitric oxide, reactive oxygen species, oxidative stress and mitochondrial transmembrane potential were significantly upregulated (P<0.01). 3. DEPs were involved in diseases and biofunctions such as cell to cell signaling and interaction, reproductive system development and function, cellular assembly and organization, protein degradation, protein synthesis, cell death and survival and cancer etc. 4. Proteins associated with key sperm functions are shown in (Table 1). 5. Proteins involved in binding of zona pellucida, binding of sperm, fertilization, spermiogenesis and spermatogenesis were downregulated and this could lead to a reduction in the fertility potential (Figure 1). 6. Downregulation of key proteins in the molecular networks can lead to asthenozoospermia, teratozoospermia, sperm disorder and infertility (Figure 2). 7. Upstream regulators such as RICTOR, IL-1a and b, IL-5, IL-6, IL-17A, LTBR, EGF, TGFB1, TGM2 etc., were activated (Z-Score ≥ 2.0). Molecular networks initiated by TFRC, alpha-Catenin, SPP1 (Osteopontin), SRF, MKL1, MKL2, XBP1, HSF1, HSF2, NFE2L2 etc., were inhibited (Z-Score ≤ 2.0) (Figure 3a-b). 8. Activation of RICTOR can lead to the down regulation of proteins involved in ubiquitination, mitochondrial dysfunction and oxidative phosphorylation (Figure 4). Figure 3. (a) The comparison analysis of the DEPs, upregulated and downregulated proteins that were activated (Z-Score ≥ 2.0) and those molecular networks that were inhibited (Z-Score ≤ 2.0) in the spermatozoa of men with SGCT compared to NC (b) Heat map showing genes in the RICTOR network Diseases or Function Annotation p-Value Down regulated Molecules # of Molecules Binding of sperm 3.48E-18 ACR, ARSA, CCT2, CCT3, CCT4, CCT5, CCT6A, CCT7, CCT8, HSPA1L, SPA17, 14 SPAM1, TCP1, ZPBP Function of sperm 0.00000337 AKAP4, ATP1A4, ATP2B4, DPY19L2, NME8, SPESP1 6 Spermatogenesis 0.00000445 ACRBP, ARSA, ATP1A4, CCT6B, DPY19L2, HSPA2, HSPA4L, NPEPPS, PSME4, 17 RUVBL1, SPA17, SPACA1, SPAG6, SPANXD (includes others), TCP1, ZPBP Fertilization 0.00000679 ACR, ACRBP, AKAP4,ARSA, C1orf56, LYZL4, SPA17, SPAM1, SPESP1, 10 Cell movement of sperm 0.0000142 AKAP4, ATP1A4, ATP2B4, NME8, PGK2, ROPN1, ROPN1B, SPAG6 8 Morphology of gonadal cells 0.0000495 ACR, AKAP4, ARSA, DPY19L2, HSPA4, PSME4, SPACA1, SPAG6, SPESP1, ZPBP 11 Abnormal morphology of acrosome 0.0000697 DPY19L2, SPACA1, SPESP1, ZPBP 4 Morphology of male germ cells 0.000135 ACR, AKAP4, DPY19L2, HSPA4, PSME4, SPACA1, SPAG6,ZPBP 8 Morphology of sperm 0.000214 ACR, AKAP4, DPY19L2, SPACA1, SPAG6 5 Morphology of germ cells 0.000332 ACR, AKAP4, DPY19L2, HSPA4, PSME4, SPACA1, SPAG6, SPESP1, ZPBP 9 Abnormal morphology of male germ cells 0.000556 ACR, AKAP4, DPY19L2, HSPA4, PSME4, SPACA1, ZPBP 7 Abnormal morphology of sperm 0.000988 ACR, AKAP4, DPY19L2, SPACA1 4 Acrosome reaction 0.000988 ACR, NSF, SLC25A1, SPESP1 4 Abnormal morphology of germ cells 0.00114 ACR, AKAP4, DPY19L2, HSPA4, PSME4, SPACA1, SPESP1, ZPBP 8 Abnormal morphology of principle piece of spermatozoon 0.00179 AKAP4, DPY19L2 2 Fertilization of sperm 0.00179 ARSA, ATP1A4 2 Penetration of Zona Pellucida 0.00179 ACR, SPAM1 2 Formation of acrosome 0.00226 DPY19L2, SPACA1, ZPBP 3 Morphology of gonad 0.00594 ACR, AKAP4, ARSA, DPY19L2, HSPA2, HSPA4, NPEPPS, PSME4, SLC2A5, 13 SPACA1, SPAG6, SPESP1 ,ZPBP Morphology of sertoli cells 0.00719 ARSA, NPEPPS, ZPBP 3 Acrosome reaction of sperm 0.00853 NSF, SLC25A1 2 Spermiogenesis 0.00935 ACRBP, DPY19L2, HSPA2, SPAG6, TCP1 5 Abnormal morphology of sperm end piece 0.00941 AKAP4 1 Differentiation of spermatocyte 0.00941 HSPA2 1 Mean curvilinear velocity of sperm 0.00941 SPAG6 1 Presence of spermatocyte 0.00941 HSPA2 1 Size of Sertoli cell 0.00941 ARSA 1 Abnormal morphology of mitochondrial sheath 0.0109 AKAP4, DPY19L2 2 Fertilization of ova 0.0123 ARSA, ATP1A4, LYZL4 3 Morphology of manchette 0.0187 ARSA, DPY19L2, HSPA4, NPEPPS, ZPBP 5 Abnormal morphology of manchette 0.0187 DYP19L2 1 Cytostasis of gonadal cell lines 0.0187 CSNK2B 1 Table 1. List of downregulated proteins involved in the reproductive system development and key spermatozoa functions in men with SGCT compared to NC Figure 4. Molecular network showing activation of RICTOR that can lead to the downregulation of proteins involved in protein ubiquitination, mitochondrial dysfunction and oxidative phosphorylation in the spermatozoa of men with SGTC. RESULTS A m e r i c a n C e n t e r f o r Rep r o d u c t i v e M e d i c i n e C e n t r u m E x c e l l e n t i a e E s t . 1 9 9 3 1. Proteins involved in the production of NO and ROS are upregulated in the spermatozoa of men with SGCT. 2. Molecular mechanism(s) of reduced fertility in these men might be attributed to the activation of up-stream regulators and the inhibition of protein networks in their spermatozoa. Nevertheless, these results merit further in-depth investigation. 3. Downregulation of essential proteins involved in spermatogenesis, spermiogenesis, acrosome reaction, sperm-oocyte binding and sperm motility function partly explains the poor sperm quality observed in SGCT patients even prior to the initiation of cancer therapy. CONCLUSIONS P-537 Figure 1. Downregulation of proteins involved in biological functions such as 1) binding of zona pellucida, 2) binding of sperm, 3) fertilization, 4) spermatogenesis and 5) spermiogenesis that can lead to a reduction in the fertility potential. Figure 2. Downregulation of key proteins in the molecular networks that can lead to 1) asthenozoospermia, 2) teratozoospermia 3) sperm disorder and 4) infertility. a. b.
Transcript of e n t e r of o r R epr d uc ti v e a n e Germ Cell Tumor ... · 1American Center for Reproductive...
1American Center for Reproductive Medicine, Urology, Cleveland Clinic, Cleveland, OH, 2Center of Excellence in Genomic Medicine Research, Jeddah, Saudi Arabia,3College of Medicine, Prince Sattam Bin Abdulaziz University, Riyadh, Saudi Arabia, 4Physiology, University of Health Sciences, Lahore, Pakistan
5Department of Urology, Cleveland Clinic, Cleveland, OH
Ashok Agarwal, PhD.1, Peter N. Pushparaj, PhD.2, Gulfam Ahmad, PhD.1,3,4, Muhammad K. Abu-Elmagd, MD.2,Mourad Assidi, PhD.2, Edmund S. Sabanegh., MD5, and Rakesh Sharma, PhD.1
Identification of Sperm Proteins Associated With Infertility in Men With Seminoma of Germ Cell Tumor Using LTQ-Orbitrap Elite Hybrid Mass Spectrometry System
ABSTRACTObjectives: Testicular germ cell tumor Seminoma (SGCT)) is the most common type of testicular cancer affecting young adult men. In these men, the semen quality begins to decline along with disturbances in the reproductive hormones. There is a lack of literature examining proteomic changes at the spermatogenic level in samples banked prior to cancer treatment. We set out to identify the sperm proteins and their role in altered semen quality in SGCT patients by proteomics and bioinformatics tool. Study Design: Identify key sperm proteins in patients with SGCT.Materials and Methods: Cryopreserved semen samples from men with SGCT (n=5) and normal healthy donors (n=5) before cancer treatment were preprocessed for proteomic analysis by LC-tandem Finnigan LTQ-Orbitrap Elite hybrid MS system. CID spectra were explored against the human reference sequence database. The relative quantity of these proteins was estimated by spectral counts (SC). The in silico examination was done using the Ingenuity Pathway Analysis (IPA) software (Qiagen, Redwood City, CA, USA) to deduce DEPs specifically linked with reproductive function. The statistical significance was calculated using Fisher’s Exact Test (P<0.05) with Benjamini-Hochberg (B-H) Multiple Testing Correction and the negative log P-value cut-off was set at 2.Results: 393 differentially expressed proteins were identified in the SGCT group. The sperm proteins associated with acrosome reaction (ACR, NSF, and SLC25A1), spermatogenesis (CCT6B, RUVBL1, SPA17, SPAG6, and SPANXD), binding of gonadal cells (ANXA5, CALR, ITGAM, ITGB2, LTF), spermiogenesis (DPY19L2, SPAG6), sperm adhesion molecule (SPAM1) involved in hyaluronic acid induced cell signaling and zona pellucida binding, sperm motility (AKAP4, ATP1A4, ROPN1B), were downregulated in SGCT group. The top differentially regulated protein network in TCS showed the downregulation of chaperons containing TCP1 subunits (CCT2, CCT3, CCT4, CCT5, CCT6A, CCT7, CCT8) that are vital for ATP-dependent folding of various proteins, including actin and tubulin in the spermatozoa.Conclusions: The proteomic analysis of semen samples in SGCT patients before cancer treatment indicates the downregulation of essential proteins involved in spermatogenesis, spermiogenesis, acrosome reaction, sperm-oocyte binding and sperm motility function. This partly explains the poor sperm quality observed in these patients even prior to the initiation of cancer therapy.
Studies have shown that more than 90 percent testicular cancers originate from germ cells. Testicular germ cell tumor Seminoma (SGCT)) is the most common type of testicular cancer affecting young adult men. SGCT is a heterogeneous type of cancer with varied histopathological and clinical manifestations. The etiology of SGCT is largely unknown. In men with SGCT, the semen quality begins to decline along with disturbances in the reproductive hormones. There is a lack of literature examining proteomic changes at the spermatogenic level in samples banked prior to cancer treatment. Here, we set out to identify the sperm proteins and their role in altered semen quality in SGCT patients by proteomics and bioinformatics tools.
INTRODUCTION
After getting institutional approval, cryopreserved semen samples from men with SGCT (n=5) and normal healthy donors (n=5) were obtained from the Andrology Biorepository for the proteomic analysis using Finnigan LTQ-Orbitrap Elite Hybrid Mass Spectrometry.
The samples were mixed with SDS Page buffer and separated on a 1D gel. Protein were digested in-gel using trypsin and peptides were extracted and evaporated to <10 µL. 30 µL was used for LC-MS analysis. Peptides eluted from the column were introduced into the source of the mass spectrometer on-line. The micro electrospray ion source is operated at 2.5 kV. The digest was analyzed using the data dependent multitask capability of the instrument. The data was analyzed, by using the CID spectra to search the human reference sequence databases with the search program Mascot and Sequest. These search results were then uploaded into the program Scaffold. The relative quantity of these proteins in the spermatozoa of SGCT patients was determined by comparing spectral counts (SC) of proteins in healthy controls.
The DEPs were then analyzed using Ingenuity Pathway Analysis (IPA) software (Qiagen, USA) to decipher the differentially regulated canonical pathways, upstream regulators, causal networks, novel networks, diseases and biofunctions. The statistical significance was calculated using Benjamini Hochberg Multiple Testing Correction (P<0.01) and the negative log P-value cut-off was fixed at 2. Besides, the comparison analyses were performed using DEPs, upregulated and down regulated proteins to further understand the proteins implicated in reduced fertility in SGCT.
MATERIALS and METHODS
1. We identified 393 proteins that were differentially regulated in the spermatozoa of men with SGCT compared to NC.2. Significantly downregulated spermatozoal proteins were implicated in the canonical pathways (P<0.01); cellular assembly and organization, cell-to-cell signaling and interaction and reproductive development and function and mitochondrial dysfunction. Proteins involved in production of nitric oxide, reactive oxygen species, oxidative stress and mitochondrial transmembrane potential were significantly upregulated (P<0.01). 3. DEPs were involved in diseases and biofunctions such as cell to cell signaling and interaction, reproductive system development and function, cellular assembly and organization, protein degradation, protein synthesis, cell death and survival and cancer etc. 4. Proteins associated with key sperm functions are shown in (Table 1). 5. Proteins involved in binding of zona pellucida, binding of sperm, fertilization, spermiogenesis and spermatogenesis were downregulated and this could lead to a reduction in the fertility potential (Figure 1). 6. Downregulation of key proteins in the molecular networks can lead to asthenozoospermia, teratozoospermia, sperm disorder and infertility (Figure 2). 7. Upstream regulators such as RICTOR, IL-1a and b, IL-5, IL-6, IL-17A, LTBR, EGF, TGFB1, TGM2 etc., were activated (Z-Score ≥ 2.0). Molecular networks initiated by TFRC, alpha-Catenin, SPP1 (Osteopontin), SRF, MKL1, MKL2, XBP1, HSF1, HSF2, NFE2L2 etc., were inhibited (Z-Score ≤ 2.0) (Figure 3a-b). 8. Activation of RICTOR can lead to the down regulation of proteins involved in ubiquitination, mitochondrial dysfunction and oxidative phosphorylation (Figure 4).
Figure 3. (a) The comparison analysis of the DEPs, upregulated and downregulated proteins that were activated (Z-Score ≥ 2.0) and those molecular networks that were inhibited (Z-Score ≤ 2.0) in the spermatozoa of men with SGCT compared to NC (b) Heat map showing genes in the RICTOR network
Diseases or Function Annotation p-Value Down regulated Molecules # of Molecules Binding of sperm 3.48E-18 ACR, ARSA, CCT2, CCT3, CCT4, CCT5, CCT6A, CCT7, CCT8, HSPA1L, SPA17, 14 SPAM1, TCP1, ZPBP Function of sperm 0.00000337 AKAP4, ATP1A4, ATP2B4, DPY19L2, NME8, SPESP1 6Spermatogenesis 0.00000445 ACRBP, ARSA, ATP1A4, CCT6B, DPY19L2, HSPA2, HSPA4L, NPEPPS, PSME4, 17 RUVBL1, SPA17, SPACA1, SPAG6, SPANXD (includes others), TCP1, ZPBP Fertilization 0.00000679 ACR, ACRBP, AKAP4,ARSA, C1orf56, LYZL4, SPA17, SPAM1, SPESP1, 10Cell movement of sperm 0.0000142 AKAP4, ATP1A4, ATP2B4, NME8, PGK2, ROPN1, ROPN1B, SPAG6 8Morphology of gonadal cells 0.0000495 ACR, AKAP4, ARSA, DPY19L2, HSPA4, PSME4, SPACA1, SPAG6, SPESP1, ZPBP 11Abnormal morphology of acrosome 0.0000697 DPY19L2, SPACA1, SPESP1, ZPBP 4Morphology of male germ cells 0.000135 ACR, AKAP4, DPY19L2, HSPA4, PSME4, SPACA1, SPAG6,ZPBP 8Morphology of sperm 0.000214 ACR, AKAP4, DPY19L2, SPACA1, SPAG6 5Morphology of germ cells 0.000332 ACR, AKAP4, DPY19L2, HSPA4, PSME4, SPACA1, SPAG6, SPESP1, ZPBP 9Abnormal morphology of male germ cells 0.000556 ACR, AKAP4, DPY19L2, HSPA4, PSME4, SPACA1, ZPBP 7Abnormal morphology of sperm 0.000988 ACR, AKAP4, DPY19L2, SPACA1 4Acrosome reaction 0.000988 ACR, NSF, SLC25A1, SPESP1 4Abnormal morphology of germ cells 0.00114 ACR, AKAP4, DPY19L2, HSPA4, PSME4, SPACA1, SPESP1, ZPBP 8Abnormal morphology of principle piece of spermatozoon 0.00179 AKAP4, DPY19L2 2Fertilization of sperm 0.00179 ARSA, ATP1A4 2Penetration of Zona Pellucida 0.00179 ACR, SPAM1 2Formation of acrosome 0.00226 DPY19L2, SPACA1, ZPBP 3Morphology of gonad 0.00594 ACR, AKAP4, ARSA, DPY19L2, HSPA2, HSPA4, NPEPPS, PSME4, SLC2A5, 13 SPACA1, SPAG6, SPESP1 ,ZPBP Morphology of sertoli cells 0.00719 ARSA, NPEPPS, ZPBP 3Acrosome reaction of sperm 0.00853 NSF, SLC25A1 2Spermiogenesis 0.00935 ACRBP, DPY19L2, HSPA2, SPAG6, TCP1 5Abnormal morphology of sperm end piece 0.00941 AKAP4 1Differentiation of spermatocyte 0.00941 HSPA2 1Mean curvilinear velocity of sperm 0.00941 SPAG6 1Presence of spermatocyte 0.00941 HSPA2 1Size of Sertoli cell 0.00941 ARSA 1Abnormal morphology of mitochondrial sheath 0.0109 AKAP4, DPY19L2 2Fertilization of ova 0.0123 ARSA, ATP1A4, LYZL4 3Morphology of manchette 0.0187 ARSA, DPY19L2, HSPA4, NPEPPS, ZPBP 5Abnormal morphology of manchette 0.0187 DYP19L2 1Cytostasis of gonadal cell lines 0.0187 CSNK2B 1
Table 1. List of downregulated proteins involved in the reproductive system development and key spermatozoa functions in men with SGCT compared to NC
Figure 4. Molecular network showing activation of RICTOR that can lead to the downregulation of proteins involved in protein ubiquitination, mitochondrial dysfunction and oxidative phosphorylation in the spermatozoa of men with SGTC.
RESULTS
Amer
ican
Center for Reproductive Medicine
Centrum Excellentiae Est.
1993
1. Proteins involved in the production of NO and ROS are upregulated in the spermatozoa of men with SGCT.2. Molecular mechanism(s) of reduced fertility in these men might be attributed to the activation of up-stream regulators and the inhibition of protein networks in their spermatozoa. Nevertheless, these results merit further in-depth investigation.3. Downregulation of essential proteins involved in spermatogenesis, spermiogenesis, acrosome reaction, sperm-oocyte binding and sperm motility function partly explains the poor sperm quality observed in SGCT patients even prior to the initiation of cancer therapy.
CONCLUSIONS
P-537
Figure 1. Downregulation of proteins involved in biological functions such as 1) binding of zona pellucida, 2) binding of sperm, 3) fertilization, 4) spermatogenesis and 5) spermiogenesis that can lead to a reduction in the fertility potential.
Figure 2. Downregulation of key proteins in the molecular networks that can lead to 1) asthenozoospermia, 2) teratozoospermia 3) sperm disorder and 4) infertility.
a. b.
