Spermatotoxic Effect of Methanol Extract of Quassia amara L.: Impact on Expression of Specific Genes Concerned with Ubiquitination-Proteosome Degradation Pathway


  • Department of Animal Science, School of Life Sciences, Bharathidasan University, Tiruchirappalli - 620024, Tamil Nadu
  • Department of Animal Science, School of Life Sciences, Bharathidasan University, Tiruchirappalli - 620024, Tamil Nadu


Defective Sperm, Proteosomal Degradation, Quassin, Quassia amara, Spermatotoxicity, Ubiquitination


Ubiquitination is believed to play a critical role in removal of dead and/or defective spermatozoa in normal and, more importantly, under circumstances when such spermatozoa are produced in large numbers due to genetic defects or toxic manifestations. Ubiquitination under such instances would involve specific gene expressions, many of which are not yet clearly understood. In an exhaustive study in Swiss mouse model to find the spermatotoxic effect of quassin, a diterpene compound isolated from Quassia amara, we found most of the spermatozoa to be abnormal in morphology and unviable. In the present study, we aimed at analysing the transcriptional profile of three selected genes, Ubb, Ube2c and Psmb8, involved in the ubiquitin proteolytic pathway in the testis and epididymal segments of Q. amara bark methanol extract treated mice adopting semi-quantitative RT-PCR and to study the level of DNA damage of the treated mouse spermatozoa. The results revealed that the treatment induced considerable damage to the sperm DNA. All the three genes studied showed marked increase in their levels of expression in the treated mice compared to the corresponding controls. Thus, this study shows that Q. amara methanol extract is causative of sperm DNA damage and defective spermatozoa and, in such cases, the expression of specific genes concerned with ubiquitination pathway is increased, implying that ubiquitination-proteosomal degradation is involved in the processing of dead/defective spermatozoa.


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Agell N, Mezquita C. Cellular content of ubiquitin and formation of ubiquitin conjugates during chicken spermatogenesis.Biochem J. 1988; 250:883–889. PMid:2839150 PMCid: PMC1148938. Available from: https://doi.org/10.1042/ bj2500883

Altamirano-Lozano M, Alvarez-Barrera L, Basurto-Alcántara F, Valverde M, Rojas E. Reprotoxic and genotoxic studies of vanadium pentoxide in male mice. Teratog Carcinog Mutage. 1996; 16:7–17. Available from: https:// doi.org/10.1002/SICI1520-6866(1996)16:1<7::AID-TCM2> 3.0.CO;2-M

Atli O, Baysal M, Aydogan-Kilic G, Kilic V, Ucarcan S, Karaduman B, Ilgin S. Sertraline-induced reproductive toxicity in male rats: evaluation of possible underlying mechanisms. Asian J Androl. 2016. DOI: 10.4103/1008682X.192637. [Epub ahead of print]. Available from: https://doi.org/10.4103/1008-682X.192637

Baarends WM, Hoogerbmgge JW, Roest HP, Qoms M, Vreeburg J, Hoeijmakers JH, Grootegoed JA. Histone ubiquitination and chromatin remodeling in mouse spermatogenesis.Develop Biol. 1999; 207:322–333. PMid:10068466.Available from: https://doi.org/10.1006/dbio.1998.9155

Baker RT, Board PG. The human ubiquitin gene family: structure of a gene and pseudogenes from the Ub B subfamily.Nucleic Acids Res. 1987l; 15:443–463. PMid:3029682 PMCid:PMC340445. Available from: https://doi.org/10.1093/nar/15.2.443

Baker RT, Board PG. The human ubiquitin-52 amino acid fusion protein gene shares several structural features with mammalian ribosomal protein genes. Nucleic Acids Res.1991; 19:1035–40. Available from: https://doi.org/10.1093/ nar/19.5.1035

Bebington C, Doherty FJ, Fleming SD. The possible biological and reproductive functions of ubiquitin. Hum Reprod Update. 2001; 7:102–11. PMid:11212067. Available from: https://doi.org/10.1093/humupd/7.1.102

BiaÅ‚y LP, Ziemba HT, Marianowski P, Fracki S, Bury M, Wójcik C. Localization of a proteasomal antigen in human spermatozoa: immunohistochemical electron microscopic study. Folia Histochem Cytobiol. 2001; 39:129–30.PMid:11374790.

Chan PJ, Corselli JU, Patton WC, Jacobson JD, Chan SR, King A. A simple comet assay for archived sperm correlates DNA fragmentation to reduced hyperactivation and penetration of zona-free hamster oocytes. Fertil Steril. 2001; 75:186–92. Available from: https://doi.org/10.1016/S00150282(00)01655-1

Chen HY, Sun JM, Zhang Y, Davie JR, Meistrich ML. Ubiquitination of histone H3 in elongating spermatids of rat testes.J Biol Chem. 1998; 273:13165–9. PMid:9582357. Available from: https://doi.org/10.1074/jbc.273.21.13165

Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987; 162:156–9. Available from: https://doi.org/10.1016/0003-2697(87)90021-2

Dong WL, Hou CC, Yang WX. Mitochondrial prohibitin and its ubiquitination during crayfish Procambarus clarkii spermiogenesis. Cell Tissue Res. 2015; 359:679–92.PMid:25418137. Available from: https://doi.org/10.1007/ s00441-014-2044-0

Donnelly ET, McClure N, Lewis SE. Glutathione and hypotaurine in vitro: Effects on human sperm motility, DNA integrity and production of reactive oxygen species. Mutagen.2000; 15: 61–8. Available from: https://doi.org/10.1093/ mutage/15.1.61

Faisal K, Girija R, Akbarsha MA. Aspects of male reproductive toxic effects of Q. amara L.: histopathological and ultrastructural study in mouse. J Endocrinol Reprod. 2015; 19:81–99.

Fatima S, Wagstaff KM, Loveland KL, Jans DA. Interactome of the negative regulator of nuclear import BRCA1-binding protein 2. Sci Rep. 2015; 5:9459. PMid:25820252 PMCid: PMC4377634. Available from: https://doi.org/10.1038/ srep09459

Finley D, Bartel B, Varshavsky A. The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis. Nature. 1989; 338: 394–401. PMid:2538753. Available from: https://doi.org/10.1038/338394a0

Fischer KA, Van Leyen K, Lovercamp KW, Manandhar G, Sutovsky M, Feng D, Safranski T, Sutovsky P. 15-Lipoxygenase is a component of the mammalian sperm cytoplasmic droplet. Reproduction. 2005; 130:213–22. PMid:16049159.Available from: https://doi.org/10.1530/rep.1.00646

Glickman MH, Ciechanover A. The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction.Physiol Rev. 2002; 82:373–428. PMid:11917093. Available from: https://doi.org/10.1152/physrev.00027.2001

Hao Y, Li R, Leng Y, Ren J, Liu J, Ai G, Xu H, Su Y, Cheng T. A study assessing the genotoxicity in rats after chronic oral exposure to a low dose of depleted uranium. J Rad Res.2009; 50:521–8. Available from: https://doi.org/10.1269/ jrr.09052

Hughes CM, Lewis SE, McKelvey-Martin VJ, Thompson W.Reproducibility of human sperm DNA measurements using the alkaline single cell gel electrophoresis assay. Mutat Res.1997; 374:261–8. Available from: https://doi.org/10.1016/ S0027-5107(96)00241-2

Jahan S, Rehman S, Ullah H, Munawar A, Ain QU, Iqbal T. Ameliorative effect of quercetin against arsenic-induced sperm DNA damage and daily sperm production in adult male rats. Drug Chem Toxicol. 2016; 39:290–6.PMid:26524343. Available from: https://doi.org/10.3109/0 1480545.2015.1101772

Jones R. Sperm survival versus degradation in the Mammalian epididymis: a hypothesis. Biol Reprod.2004; 71:1405–11. PMid:15215193. Available from: https://doi.org/10.1095/biolreprod.104.031252

Kay GF, Ashworth A, Penny GD, Dunlop M, Swift S, Brockdorff N, Rastan S. A candidate spermatogenesis gene on the mouse Y chromosome is homologous to ubiquitin-activating enzyme E1. Nature. 1991; 354:486–9. PMid:1749428.Available from: https://doi.org/10.1038/354486a0

Kerns K, Morales P, Sutovsky P. Regulation of Sperm Capacitation by the 26S Proteasome: An Emerging New Paradigm in Spermatology. Biol Reprod. 2016; 94:117.PMid:27053366. Available from: https://doi.org/10.1095/ biolreprod.115.136622

Koenig PA, Nicholls PK, Schmidt FI, Hagiwara M, Maruyama T, Frydman GH, Watson N, Page DC, Ploegh HL. The E2 ubiquitin-conjugating enzyme UBE2J1 is required for spermiogenesis in mice. J Biol Chem. 2014; 289:34490–502. PMid:25320092 PMCid:PMC4263858.Available from: https://doi.org/10.1074/jbc.M114.604132

KoÅ„ca K, Lankoff A, Banasik A, Lisowska H, Kuszewski T, Góźdź S, Koza Z, Wojcik A. A cross-platform public domain PC image-analysis program for the comet assay.Mutat Res. 2003; 534:15–20. Available from: https://doi.org/10.1016/S1383-5718(02)00251-6

Lehrach H, Diamond D, Wozney JM, Boedtker H. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry.1977; 16:4743–4751. PMid:911786. Available from: https://doi.org/10.1021/bi00640a033

Lippert TH, Seeger H, Schieferstein G, Voelter W. Immunoreactive ubiquitin in human seminal plasma. J Androl.1993; 14:130–131. PMid:8390427.

Lund PK, Moats-Staats BM, Simmons JG, Hoyt E, D'Ercole AJ, Martin F, Van Wyk JJ. Nucleotide sequence analysis of a cDNA encoding human ubiquitin reveals that ubiquitin is synthesized as a precursor. J Biol Chem. 1985; 260:7609–13.PMid:2581967.

Luo M, Zhou J, Leu NA, Abreu CM, Wang J, Anguera MC, de Rooij DG, Jasin M, Wang PJ. Polycomb protein SCML2 associates with USP7 and counteracts histone H2A ubiquitination in the XY chromatin during male meiosis. PLoS Genet. 2015; 11:e1004954. Available from: https://doi.org/10.1371/journal.pgen.1004954

Ma T, Keller JA, Yu X. RNF8-dependent histone ubiquitination during DNA damage response and spermatogenesis.Acta Biochem Biophys Sin (Shanghai). 2011; 43:339– 45. PMid:21444325 PMCid:PMC3080603. Available from: https://doi.org/10.1093/abbs/gmr016

Mochida K, Tres LL, Kierszenbaum AL. Structural features of the 26S proteasome complex isolated from rat testis and sperm tail. Mol Reprod Dev. 2000; 57:176–84. Available from: https://doi.org/10.1002/10982795(200010)57:2<176::AID-MRD9>3.0.CO;2-O

Mtango NR, Latham KE. Ubiquitin proteasome pathway gene expression varies in rhesus monkey oocytes and embryos of different developmental potential. Physiol Genom.2007; 31:1–14. PMid:17550997. Available from: https://doi.org/10.1152/physiolgenomics.00040.2007

Nandi D, Tahiliani P, Kumar A, Chandu D. The ubiquitinproteasome system. J Biosci. 2006; 31:137–55.PMid:16595883. Available from: https://doi.org/10.1007/ BF02705243

Oakberg EF. Duration of spermatogenesis in the mouse and timing of stages of the cycle of the seminiferous epithelium.Am J Anat. 1956; 99:507–16. PMid:13402729. Available from: https://doi.org/10.1002/aja.1000990307

Rajapurohitam V, Morales CR, El-Alfy M, Lefrançois S, Bedard N, Wing SS. Activation of a UBC4-dependent pathway of ubiquitin conjugation during postnatal development of the rat testis. Dev Biol. 1999; 212:217–28. PMid:10419697.Available from: https://doi.org/10.1006/dbio.1999.9342

Rajapurohitam V, Bedard N, Wing SS. Control of ubiquitination of proteins in rat tissues by ubiquitin conjugating enzymes and isopeptidases. Am J Physiol Endocrinol Metabol.2002; 282:739–45. PMid:11882492. Available from: https://doi.org/10.1152/ajpendo.00511.2001

Redman KL, Rechsteiner M. Identification of the long ubiquitin extension as ribosomal protein S27a. Nature.1989; 338:438–40. PMid:2538756. Available from: https://doi.org/10.1038/338438a0

Rivett AJ, Hearn AR. Proteasome function in antigen presentation: immunoproteasome complexes, Peptide production, and interactions with viral proteins. Curr Prot Pept Sci. 2004; 5:153–61. Available from: https://doi.org/10.2174/1389203043379774

Ryu KY, Maehr R, Gilchrist CA, Long MA, Bouley DM, Mueller B, Ploegh HL, Kopito RR. The mouse polyubiquitin gene UbC is essential for fetal liver development, cell-cycle progression and stress tolerance. EMBO J. 2007; 26:2693– 706. PMid:17491588 PMCid:PMC1888680. Available from: https://doi.org/10.1038/sj.emboj.7601722

Ryu KY, Sinnar SA, Reinholdt LG, Vaccari S, Hall S, Garcia MA, Zaitseva TS, Bouley DM, Boekelheide K, Handel MA, Conti M, Kopito RR. The mouse polyubiquitin gene Ubb is essential for meiotic progression. Mol Cell Biol. 2008; 28:1136–46. PMid:18070917 PMCid:PMC2223379. Available from: https://doi.org/10.1128/MCB.01566-07

Sakkas D, Mariethoz E, Manicardi G, Bizzaro D, Bianchi PG, Bianchi U. Origin of DNA damage in ejaculated human spermatozoa. Rev Reprod. 1999; 4:31–7. PMid:10051100.Available from: https://doi.org/10.1530/ror.0.0040031

Serafini R, Love CC, Coletta A, Mari G, Mislei B, Caso C, Di Palo R. Sperm DNA integrity in frozen-thawed semen from Italian Mediterranean Buffalo bulls and its relationship to in vivo fertility. Animal Reprod Sci. 2016; 172:26–31.PMid:27421229. Available from: https://doi.org/10.1016/j.anireprosci.2016.06.010

Sharma V, Shukla RK, Saxena N, Parmar D, Das M, Dhawan A. DNA damaging potential of zinc oxide nanoparticles in human epidermal cells. Toxicol Lett. 2009; 185:211–218.PMid:19382294. Available from: https://doi.org/10.1016/j.toxlet.2009.01.008

Sheng K, Liang X, Huang S, Xu W. The role of histone ubiquitination during spermatogenesis. Biomed Res Int. 2014. DOI: 10.1155/2014/870695. Available from: https://doi.org/10.1155/2014/870695

Singh NP, Stephens RE. X-ray induced DNA double-strand breaks in human sperm. Mutagenesis. 1998; 13:75–9. Available from: https://doi.org/10.1093/mutage/13.1.75

Singh S, Awasthi N, Egwuagu CE, Wagner BJ. Immunoproteasome expression in a nonimmune tissue, the ocular lens.Arch Biochem Biophys. 2002; 405:147–53. Available from: https://doi.org/10.1016/S0003-9861(02)00341-7

Hikim APS, Swerdloff RS. Hormonal and genetic control of germ cell apoptosis in the testis. Rev Reprod. 1999; 4:38–47.Available from: https://doi.org/10.1530/ror.0.0040038

Song WH, Ballard JW, Yi YJ, Sutovsky P. Regulation of mitochondrial genome inheritance by autophagy and ubiquitin-proteasome system: implications for health, fitness, and fertility. Biomed Res Int. 2014. doi.org/10.1155/2014/981867. Available from: https://doi.org/10.1155/2014/981867

Steele EK, McClure N, Lewis SE. Comparison of the effects of two methods of cryopreservation on testicular sperm DNA. Fertil Steril. 2000; 74:450–3. Available from: https:// doi.org/10.1016/S0015-0282(00)00680-4

Strous GJ, Govers R. The ubiquitin-proteasome system and endocytosis. J Cell Sci. 1999; 112:1417–23. PMid:10212136.

Sutovsky P. Ubiquitin-dependent proteolysis in mammalian spermatogenesis, fertilization, and sperm quality control: killing three birds with one stone. Micros Res Tech.2003; 61:88–102. PMid:12672125. Available from: https:// doi.org/10.1002/jemt.10319

Sutovsky P. Sperm proteasome and fertilization. Reproduction.2011; 142:1–14. PMid:21606061. Available from: https://doi.org/10.1530/REP-11-0041

Sutovsky P, Moreno RD, Ramalho-Santos J, Dominko T, Simerly C, Schatten G. Ubiquitin tag for sperm mitochondria.Nature. 1999; 402:371–2. PMid:10586873. Available from: https://doi.org/10.1038/46466

Sutovsky P, Moreno RD, Ramalho-Santos J, Dominko T, Simerly C, Schatten G. Ubiquitinated sperm mitochondria, selective proteolysis, and the regulation of mitochondrial inheritance in mammalian embryos. Biol Reprod. 2000; 63:582–90. PMid:10906068. Available from: https://doi.org/10.1095/biolreprod63.2.582

Sutovsky P, Moreno R, Ramalho-Santos J, Dominko T, Thompson WE, Schatten G. A putative, ubiquitin-dependent mechanism for the recognition and elimination of defective spermatozoa in the mammalian epididymis. J Cell Sci. 2001a; 114:1665–75. PMid:11309198.

Sutovsky P, Terada Y, Schatten G. Ubiquitin-based sperm assay for the diagnosis of male factor infertility. Hum Reprod.2001b; 16:250–8. PMid:11157815. Available from: https://doi.org/10.1093/humrep/16.2.250

Sutovsky P, Neuber E, Schatten G. Ubiquitin-dependent sperm quality control mechanism recognizes spermatozoa with DNA defects as revealed by dual ubiquitin-TUNEL assay. Mol Reprod Dev. 2002; 61:406–13. PMid:11835586.Available from: https://doi.org/10.1002/mrd.10101

Sutovsky P, Hauser R, Sutovsky M. Increased levels of sperm ubiquitin correlate with semen quality in men from an andrology laboratory clinic population. Hum Reprod.2004; 19:628–38. PMid:14998962. Available from: https:// doi.org/10.1093/humrep/deh131

Tengowski MW, Sutovsky P, Hedlund LW, Guyot DJ, Burkhardt JE, Thompson WE, Sutovsky M, Johnson GA. Reproductive cytotoxicity is predicted by magnetic resonance microscopy and confirmed by ubiquitinproteasome immunohistochemistry in a theophylline-induced model of rat testicular and epididymal toxicity. Microsc Microanal.2005; 11:300–12. PMid:16079014. Available from: https:// doi.org/10.1017/S143192760505021X

Tengowski MW, Feng D, Sutovsky M, Sutovsky P. Differential expression of genes encoding constitutive and inducible 20S proteasomal core subunits in the testis and epididymis of theophylline- or 1,3-dinitrobenzene-exposed rats. Biol Reprod. 2007; 76:149–63. PMid:16988215. Available from: https://doi.org/10.1095/biolreprod.106.053173

Tipler CP, Hutchon SP, Hendil K, Tanaka K, Fishel S, Mayer RJ. Purification and characterization of 26S proteasomes from human and mouse spermatozoa. Mol Hum Reprod.1997; 3:1053–60. PMid:9464850. Available from: https:// doi.org/10.1093/molehr/3.12.1053

Wiborg O, Pedersen MS, Wind A, Berglund LE, Marcker KA, Vuust J. The human ubiquitin multigene family: some genes contain multiple directly repeated ubiquitin coding sequences. EMBO J. 1985; 4:755–9. PMid:2988935 PMCid: PMC554252.

Wing SS, Jain P. Molecular cloning, expression and characterization of a ubiquitin conjugation enzyme (E2(17)kB)highly expressed in rat testis. Biochem J. 1995; 305:125–32.PMid:7826319 PMCid:PMC1136439. Available from: https://doi.org/10.1042/bj3050125

Wing SS, Bédard N, Morales C, Hingamp P, Trasler J. A novel rat homolog of the Saccharomyces cerevisiae ubiquitinconjugating enzymes UBC4 and UBC5 with distinct biochemical features is induced during spermatogenesis.Mol Cell Biol. 1996; 16:4064–72. PMid:8754804 PMCid: PMC231402. Available from: https://doi.org/10.1128/ MCB.16.8.4064

Wojcik C, Benchaib M, Lornage J, Czyba JC, Guerin JF. Proteasomes in human spermatozoa. Int J Androl.2000; 23:169–77. PMid:10844543. Available from: https://doi.org/10.1046/j.1365-2605.2000.00223.x




How to Cite

Faisal, K., & Akbarsha, M. A. (2017). Spermatotoxic Effect of Methanol Extract of Quassia amara L.: Impact on Expression of Specific Genes Concerned with Ubiquitination-Proteosome Degradation Pathway. Journal of Endocrinology and Reproduction, 20(1), 55–65. Retrieved from https://informaticsjournals.com/index.php/jer/article/view/15686




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