RNA Sequencing Data Analysis of Oligoasthenoteratozoospermia Patients' Sperms and Effects of Curcumin on the Expression of Some Genes during Sperm Cryopreservation and Freeze-Thawing Process
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Abstract
Background: The cause of oligoasthenoteratozoospermia (OAT) is unclear. In this original study, we examined OAT patients' sperm using RNA-sequencing and studied the impact of curcumin on gene expression during sperm cryopreservation and freeze-thawing.
Methods: RNA-seq was performed using the Galaxy Europe server in IKHC (Imam Khomeini Hospital Complex), Tehran, Iran in 2023. Sperm samples were collected from 30 OAT patients and 30 healthy volunteers. Sperm parameters were analyzed, and samples were frozen with 20 μM curcumin at -196 °C for one week. Thawed samples were assessed for sperm parameters, and the expression levels of bax, bak, bcl-2, bclw, casp9, apaf-1, SOD, cat, and GPX4 genes were measured using RT-PCR.
Result: RNA-seq analysis showed increased expression of NANOS1, HSPA6, and ALOXE3 genes, while BHLHE41, Hey1, and PPM1D genes were down-regulated in OAT patients' sperm. Curcumin (20 μM) effectively preserves sperm parameters and motility during cryopreservation in healthy subjects and in OAT patients in particular. Cryopreserved sperm from both OAT patients and healthy individuals exhibited reduced expression of pro-apoptotic genes, increased expression of anti-apoptotic genes, and elevated levels of SOD and GPX4 genes.
Conclusion: Altered expressions of NANOS1, HSPA6, ALOXE3, BHLHE41, Hey1, and PPM1D genes likely contribute to OAT development. Additionally, curcumin protects sperm parameters during cryopreservation for both healthy individuals and OAT patients.
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12. Santonastaso M, Mottola F, Iovine C, et al (2021). Protective Effects of Curcumin on the Outcome of Cryopreservation in Human Sperm. Reprod Sci, 28(10):2895-2905.
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14. Janecki DM, Ilaslan E, Smialek MJ, et al (2019). A male infertility mutation re-verts NANOS1 activity from anti-apoptotic to pro-apoptotic by disrupting repression of GADD45A, GADD45B, GADD45G and RHOB genes. bioRxiv, 28(3):858-869.
15. Ginter-Matuszewska B, Kusz K, Spik A, et al (2011). NANOS1 and PUMILIO2 bind microRNA biogenesis factor GEMIN3, within chromatoid body in human germ cells. Histochem Cell Biol, 136(3):279-287.
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17. Haraguchi S, Tsuda M, Kitajima S, et al (2003). NONOS1 a mouse nanos gene expressed in the central nervous system is dispensable for normal development. Mech Dev, 120(6):721-731.
18. Janecki DM, Ilaslan E, Smialek MJ, et al (2020). Human NANOS1 Represses Apoptosis by Downregulating Pro-Apoptotic Genes in the Male Germ Cell Line. Int J Mol Sci, 21(8):3009.
19. Leung T, Rajendran M, Monfries C, et al (1990). The human heat-shock protein family, Expression of a novel heat-inducible HSP70 (HSP70B′) and isolation of its cDNA and genomic DNA. Biochem J, 267(1):125-132.
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21. Sun Y, Xue Z, Huang T, et al (2022). Lipid metabolism in ferroptosis and ferropto-sis-based cancer therapy. Front Oncol, 12:941618
22. Kreslavsky T, Vilagos B, Tagoh H, et al (2017). Essential role for the transcrip-tion factor Bhlhe41 in regulating the de-velopment, self-renewal and BCR reper-toire of B-1a cells. Nat Immunol, 18(4):442-455.
23. Shen Z, Zhu L, Zhang C, et al (2019). Over-expression of BHLHE41, correlated with DNA hypomethylation in 3'UTR region, promotes the growth of human clear cell renal cell carcinoma. Oncol Rep, 41(4):2137-2147.
24. Van Wayenbergh R, Taelman V, Pichon B, F et al (2003). Identification of BOIP, a novel cDNA highly expressed during spermatogenesis that encodes a protein interacting with the orange domain of the hairy-related transcription factor HRT1/Hey1 in Xenopus and mouse. Dev Dyn, 228(4):716-725.
25. Chuman Y, Kurihashi W, Mizukami Y, et al (2009).A novel alternative splicing vari-ant of the human PPM1D, can dephosphorylate p53 and exhibits spe-cific tissue expression. J Biochem, 145(1):1-12.
26. Zhang X, Lu X, Li J, et al (2019). Mito-Tempo alleviates cryodamage by regu-lating intracellular oxidative metabolism in spermatozoa from asthenozoosper-mic patients. Cryobiology, 91:18-22.
27. Rezaeian Z, Yazdekhasti H, Nasri S, et al (2016). Effect of selenium on human sperm parameters after freezing and thawing procedures. Asian Pac J Reprod, 5(6):462-466.
28. Kopeika J, Thornhill A, Khalaf Y (2015). The effect of cryopreservation on the genome of gametes and embryos: prin-ciples of cryobiology and critical ap-praisal of the evidence. Hum Reprod Up-date, 21(2):209-227.
29. Bratton SB, Walker G, Srinivasula SM, et al (2001). Recruitment, activation and re-tention of caspases-9 and -3 by Apaf-1 apoptosome and associated XIAP com-plexes. EMBO J, 20(5):998-1009.
30. Zhou M, Li Y, Hu Q, et al (2015). Atomic structure of the apoptosome: mecha-nism of cytochrome c-and dATP-mediated activation of Apaf-1. Genes Dev, 29(22):2349-2361.
31. Shimizu S, Narita M, Tsujimoto Y, et al (1999). Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC. Na-ture, 399(6735):483-487.
32. Lomonosova E, Chinnadurai G (2008). BH3-only proteins in apoptosis and be-yond: an overview. Oncogene, 27 Suppl 1(Suppl 1):S2-19.
2. Okonofua FE, Ntoimo LFC, Omonkhua A, et al (2022). Causes and Risk Factors for Male Infertility. Int J Gen Med, 15:5985-5997.
3. Herwig R, Knoll C, Planyavsky M, et al (2013). Proteomic analysis of seminal plasma from infertile patients with oli-goasthenoteratozoospermia due to oxi-dative stress and comparison with fertile volunteers. Fertil Steril, 100(2):355-366.e2.
4. Oehninger S (2003). Pathophysiology of ol-igoasthenoteratozoo-spermia are we improving in the diagnosis. Reprod Bio-med Online, 7(4):433-439.
5. Weng H-Y, Lin T-Y, Lin Y-M, et al (2021). The fertility preservation decision-making and testicular sperm retrieval outcome in older adolescents with nonmosaic Klinefelter syndrome and azoospermia. J Chin Med Assoc, 84(11):1023-1027.
6. Khan IM, Cao Z, Liu H, et al (2021). Impact of Cryopreservation on Spermatozoa Freeze-Thawed Traits and Relevance OMICS to Assess Sperm Cryo-Tolerance in Farm Animals. Front Vet Sci, 8: 609180.
7. Yurchuk T, Petrushkо M, Gapon A, et al (2021). The impact of cryopreservation on the morphology of spermatozoa in men with oligoasthenoteratozoo-spermia. Cryobiology, 100 :117-124.
8. Cankut S, Dinc T, Cincik M, et al (2019). Evaluation of sperm DNA fragmenta-tion via halosperm technique and TUNEL assay before and after cryo-preservation. Reprod Sci, 26(12):1575-1581.
9. Shaliutina-Loginova A, Loginov DS (2023). Oxidative stress and DNA fragmenta-tion in frozen/thawed common Carp Cyprinus carpio sperm with and without supplemental proteins. Anim Reprod Sci, 251:107213.
10. Brugnon F, Ouchchane L, Artonne C, et al (2013). Density gradient centrifugation prior to cryopreservation and anti-oxydant supplementation by hypotau-rine improve post-thaw sperm quality of infertile men with oligoasthenoterato-zoospermia. Hum Reprod, 28(8):2045-57.
11. Nur Karakus F, Bulgurcuoglu Kuran S, Solakoglu S (2021). Effect of curcumin on sperm parameters after the cryo-preservation. Eur J Obstet Gynecol Reprod Biol, 267:161-166.
12. Santonastaso M, Mottola F, Iovine C, et al (2021). Protective Effects of Curcumin on the Outcome of Cryopreservation in Human Sperm. Reprod Sci, 28(10):2895-2905.
13. Tvrdá E, Greifová H, Mackovich A, et al (2018). Curcumin offers antioxidant pro-tection to cryopreserved bovine semen. Czech J Anim Sci, 63(7):247-255.
14. Janecki DM, Ilaslan E, Smialek MJ, et al (2019). A male infertility mutation re-verts NANOS1 activity from anti-apoptotic to pro-apoptotic by disrupting repression of GADD45A, GADD45B, GADD45G and RHOB genes. bioRxiv, 28(3):858-869.
15. Ginter-Matuszewska B, Kusz K, Spik A, et al (2011). NANOS1 and PUMILIO2 bind microRNA biogenesis factor GEMIN3, within chromatoid body in human germ cells. Histochem Cell Biol, 136(3):279-287.
16. Kusz-Zamelczyk K, Sajek M, Spik A, et al (2013). Mutations of NANOS1, a human homologue of the Drosophila morpho-gen, are associated with a lack of germ cells in testes or severe oligo-astheno-teratozoospermia. J Med Genet, 50(3):187-193.
17. Haraguchi S, Tsuda M, Kitajima S, et al (2003). NONOS1 a mouse nanos gene expressed in the central nervous system is dispensable for normal development. Mech Dev, 120(6):721-731.
18. Janecki DM, Ilaslan E, Smialek MJ, et al (2020). Human NANOS1 Represses Apoptosis by Downregulating Pro-Apoptotic Genes in the Male Germ Cell Line. Int J Mol Sci, 21(8):3009.
19. Leung T, Rajendran M, Monfries C, et al (1990). The human heat-shock protein family, Expression of a novel heat-inducible HSP70 (HSP70B′) and isolation of its cDNA and genomic DNA. Biochem J, 267(1):125-132.
20. Deane CA, Brown IR (2018). Knockdown of heat shock proteins HSPA6 (Hsp70B’) and HSPA1A (Hsp70-1) sensitizes differ-entiated human neuronal cells to cellu-lar stress. Neurochem Res, 43(2):340-350.
21. Sun Y, Xue Z, Huang T, et al (2022). Lipid metabolism in ferroptosis and ferropto-sis-based cancer therapy. Front Oncol, 12:941618
22. Kreslavsky T, Vilagos B, Tagoh H, et al (2017). Essential role for the transcrip-tion factor Bhlhe41 in regulating the de-velopment, self-renewal and BCR reper-toire of B-1a cells. Nat Immunol, 18(4):442-455.
23. Shen Z, Zhu L, Zhang C, et al (2019). Over-expression of BHLHE41, correlated with DNA hypomethylation in 3'UTR region, promotes the growth of human clear cell renal cell carcinoma. Oncol Rep, 41(4):2137-2147.
24. Van Wayenbergh R, Taelman V, Pichon B, F et al (2003). Identification of BOIP, a novel cDNA highly expressed during spermatogenesis that encodes a protein interacting with the orange domain of the hairy-related transcription factor HRT1/Hey1 in Xenopus and mouse. Dev Dyn, 228(4):716-725.
25. Chuman Y, Kurihashi W, Mizukami Y, et al (2009).A novel alternative splicing vari-ant of the human PPM1D, can dephosphorylate p53 and exhibits spe-cific tissue expression. J Biochem, 145(1):1-12.
26. Zhang X, Lu X, Li J, et al (2019). Mito-Tempo alleviates cryodamage by regu-lating intracellular oxidative metabolism in spermatozoa from asthenozoosper-mic patients. Cryobiology, 91:18-22.
27. Rezaeian Z, Yazdekhasti H, Nasri S, et al (2016). Effect of selenium on human sperm parameters after freezing and thawing procedures. Asian Pac J Reprod, 5(6):462-466.
28. Kopeika J, Thornhill A, Khalaf Y (2015). The effect of cryopreservation on the genome of gametes and embryos: prin-ciples of cryobiology and critical ap-praisal of the evidence. Hum Reprod Up-date, 21(2):209-227.
29. Bratton SB, Walker G, Srinivasula SM, et al (2001). Recruitment, activation and re-tention of caspases-9 and -3 by Apaf-1 apoptosome and associated XIAP com-plexes. EMBO J, 20(5):998-1009.
30. Zhou M, Li Y, Hu Q, et al (2015). Atomic structure of the apoptosome: mecha-nism of cytochrome c-and dATP-mediated activation of Apaf-1. Genes Dev, 29(22):2349-2361.
31. Shimizu S, Narita M, Tsujimoto Y, et al (1999). Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC. Na-ture, 399(6735):483-487.
32. Lomonosova E, Chinnadurai G (2008). BH3-only proteins in apoptosis and be-yond: an overview. Oncogene, 27 Suppl 1(Suppl 1):S2-19.
| Files | ||
| Issue | Vol 54 No 1 (2025) | |
| Section | Original Article(s) | |
| Keywords | ||
| Oligoasthenoteratozoospermia Sperm Cryopreservation RNA sequencing Curcumin | ||
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How to Cite
1.
Naseri F, Siasy E, Angaji SA, Peyvandi M. RNA Sequencing Data Analysis of Oligoasthenoteratozoospermia Patients’ Sperms and Effects of Curcumin on the Expression of Some Genes during Sperm Cryopreservation and Freeze-Thawing Process. Iran J Public Health. 2025;54(1):214-224.
