1. Molecular Dissection of Events Leading to Varicocele Induced Male Infertility Kothandaraman Narasimhan, Ralf Henkel Department of Medical Biosciences, University of the Western Cape, Bellville, South Africa
Figure 6. Mitochondrial dysfunction plays a major role in the pathogenesis and aggravation of VC. TP53 and BCL2 regulate mitochondrial membrane permeabilization involved in germ cell apoptosis. This is initiated by mitochondrial Cyt c release activated through cysteine-type endopeptidase activity. This process compromises mitochondrial genome stability through damage caused by faulty base-excision repair. The above events leads to death of germ and epithelial cells surrounding VC tissues resulting in azoospermia by affecting genes MEI1 and ING2.
Introduction:
Varicocele (VC) is a pathological condition resulting in male infertility (MI). We used a gene-based approach to reconstruct the intrinsic networks involved in VC pathogenesis leading to MI. Gene signatures unique to each etiological factor that leads to the progression of VC are elucidated in the current study.
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Figure 7. Methylation resulting in changes in the epigenome leading to abnormalities associated with MI. Methylation mostly happens through inter-ference from ET factors on sperm heterochromatin. SNRPN, H19 and MTHFR genes were identified to be associated with methylation in VC. Methylation specifically targets heterochromatin maintenance and MTHFR promoter as shown for OAT conditions in VC patients. Similarly genetic imprinting and loss of methylation at the H19 imprinted gene correlates with MTHF hypermethylation in infertile male patients.
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Material and Methods:
For the purpose of constructing gene-based networks and pathways, we manually curated gene sets that were reported to be associated with VC and MI. A total of more than 100 genes were catalogued that showed strong links with the events leading to the pathogenesis of VC induced MI (Fig. 1). Using network analysis, key nodal genes as well as pathways were reconstructed. This process helped in the identification of the missing links involved in connecting distinctly unrelated pathways.
Figure 8. Ca2+-channel proteins are required for maintaining homeostasis of ion fluxes during cell development related to spermatogenesis. The CATSPER gene is similar to voltage-dependent Ca2+-channel α1 subunit and among the most character-rized channel genes associated with sperm develop-ment. VC induces potential membrane depolarization resulting in the development of abnormal sperm and impaired motility. Phospholipase activity has a role in testosterone-mediated activation of phospholipase C and Ca2+-influx into Sertoli cells which is required for normal spermatogenesis. The intermediate linking genes that connect these two pathways were identified as PRKACB and PRKACG, respectively.
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Results:
Figure 1. Etiological factors in VC pathogenesis. This study used at least 9 key etiological factors that could result in the development of VC-induced MI (each segment in the diagram indicates the percentage of genes). Conditions involving oxidative stress (OS), reactive oxygen species metabolism (ROS), hypoxic environment, methylation, higher rate of DNA damage, Ca2+-channel abnormalities, and genes coded by Y-chromosome were found to contribute to VC pathogenesis. Mitochondria play a central role in the network and are associated with a cascade of events resulting in VC-induced MI phenotype.
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Figure 9. Hypoxic condition induced by VC initiates RNA polymerase II promoter transcription in response to stress. Key genes involved in this process are HIF1A, NOTCH1 and tumour suppressor gene TP53. Hypoxic conditions in VC also stem inflammatory response similar to antigenic stimulus through AK7 and NOTCH genes. Our analysis shows stress response induced by hypoxia as well as inflammation triggered by antigenic stimulus contributes to the pathogenesis of VC.
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Figure 2. Vessel morphogenesis in VC. Aberrant insulin and hexose metabolic process affect blood vessel morphogenesis, size and angiogenesis in VC pathogenesis. Insulin and hexose metabolism genes affects processes such as vasodilation, prostate and Sertoli cell differentiation. INS and ESR1 are involved in regulation of NO2 metabolic process and vasodilation. Key genes in this process include INS, GNRH1, ESR, IGF1 and CD4. The above processes affect normal spermatogenesis.
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Figure 10. Y chromosome and VC. Three genes USP9Y, CFTR, DAZ1 were associated with Y chromosome.
Figure 11. CFTR gene coded by Y-chromosome is primarily involved with membrane hyperpolarization. Sperm capacitation is primarily associated with membrane permeability and is carried out by CFTR gene. DAZ1 functions as positive regulation of translation initiation. Through our analysis we also identified both activators and inhibitors of CFTR1.
Conclusions:
Based on systems biology platform using network analysis, we identified more than 100 genes which contribute to VC through different pathways and signaling events. The results indicate a multifactorial set of complex events compounded by extraneous factors leading to VC-induced MI. Hidden links from several disjointed studies were integrated to identify the disease pathways that lead to the progression of the disease. OS and ROS generation as well as mitochondrial membrane permeability were found to be the major contributory factors for VC pathogenesis. Environmental factors trigger OS through production of ROS leading to DNA damage triggering apoptosis through TP53 pathways in VC patients. These events lead to significant damage to germ cells and mature/immature sperm. Role of methylation and hypoxia showed candidate marker genes that could help in the prognosis and treatment for this disease condition.
Figure 3. OS affects spermatogenesis. DNA instability as well as apoptosis are direct effects of OS that leads to MI in VC patients. OS triggers ROS as well as targets mitochondrial metabolism through JAK-STAT pathway. Several genes contribute to this condition.
Figure 4. Generation of ROS leads to nucleotide catabolic process resulting in DNA damage. p38MAPK pathway is activated. ROS also damage lipid rafts resulting in aberrant cell proliferation.
Figure 5. Environmental toxicants (ET) correlate with gene expression in VC patients. Exposure to ETs results in increased OS and generate ROS molecules. Both these process directly affect spermatogenesis. One visible effect of ETs is oligozoospermic condition in VC patients due to abnormal FSHB, RBMY1A1, SOD1, and PARP1 gene expression. ET primarily cause apoptosis through TP53 pathway through combination of factors resulting in cell death in VC patients resulting in synthesis of nonviable male gametes.
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