Effect of nimbolide on rat spermatozoa under in-vitro condition: A study on sperm functional events and antioxidants during capacitation
DOI:
https://doi.org/10.48165/jlas.2024.7.1.3Keywords:
Nimbolide, Spermatozoa, Motility, ATP content, Biochemical, AR and RatAbstract
However, little evidence has been documented to evaluate the clear specific effects of neem-active ingredients in a rat model. This study aims to evaluate the in vitro effects of nimbolide (tetranortriterpenoids group, one of the main components of neem leaves), particularly on sperm functional studies, and biochemical and molecular events during capacitation in a dose dependent manner. The results showed a dose- and time-dependent decrease in the functional consequence of capacitation process i.e., motility score, percentage of motile spermatozoa, Sperm Motility Index (SMI) and levels of molecular events in sperm followed by declined spontaneous acrosome reaction (AR), which subsequently leads to lesser binding of cauda epididymal sperm to the Zona pellucida (ZP). Biochemical studies have shown that the activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase, Glutathione reductase (GR) and Glutathione peroxidase (GPx) decreased significantly while the levels of Hydrogen peroxide (H2O2) generation and lipid peroxidation (LPO) increased significantly in the treated groups indicating nimbolide-induced oxidative stress in rat epididymal sperm. This study confirmed that nimbolide convincingly inhibited sperm motility in rats by blocking certain biochemical pathways, such as energy utilization, and showed that sperm capacitation was associated with a reduction in AR, with changes in antioxidant enzymes reflecting a reduction at the levels of molecular events. This will lead us to produce a special product as a source of a new male contraceptive.
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References
1. Agarwal A, Sharma R, Gupta S, Harlev A, Ahmad G, du Plessis SS, Esteves SC, Wang SM, Durairajanayagam D (2017). Oxidative stress in human reproduction: shedding light on a complicated phenomenon. eds. New York City, NY, USA: Springer Publishing.
2. Ain R, Uma Devi K, Shivaji S, Seshagiri PB (1999). Pentoxifylline-stimulated capacitation and acrosome reaction in hamster spermatozoa: involvement of intracellular signalling molecules. Mol Hum Reprod. 57:618-626.
3. Aitken RJ, Harkiss D, Knox W, Paterson M, Irvine DS (1998). A novel signal transduction cascade in capacitating human spermatozoa characterized by a redox-regulated, camp mediated induction of tyrosine phosphorylation. J Cell Sci. 111:645-656.
4. Aitken RJ, Paterson M, Fisher H, Buckingham DW, van Duin M (1995). Redox regulation of tyrosine phosphorylation in human spermatozoa and its role in the control of human sperm function. J Cell Sci.108:2017-
2025.
5. Aladakatti RH, Sukesh B, Jadaramkunti UC, Hiremath M (2011). Effect of graded doses of nimbolide (a major component of Azadirachta indica leaves) on biochemical and sperm functional parameters in male albino rats. J LabRes. 8(5):445–449.
7. Baligar NS, Aladakatti RH, Mukhtar Ahmed, Hiremath MB (2014). Hepatoprotective activity of neem based constituent azadirachtin-A in carbon tetrachloride intoxicated albino rats. Can J Physiol Pharmacol. 92: 267-277.
8. Baskaran S, Finelli R, Agarwal A, Henkel, R (2021). Reactive oxygen species in male reproduction: A boon or a bane? Andrologia.53: e13577.
9. Bavister BD, Andrews JC (1988). A rapid sperm motility bioassay procedure for quality- control testing of water and culture media. J In Vitro Fert Embryo Transf. 52:67- 75.
10. Beltran C, Vacquier VD, Moy G, Chen Y, Buck J, Levin LD, Darszon A (2007). Particulate and soluble adenylyl cyclases participate in the sperm acrosome reaction. Biochem Biophys Res Commun. 358:1128–1135.
11. Burton K (1956). A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. J Biochem. 62: 316– 320.
12. Carlberg I, Mannervik BJ (1985). Purification and characterization of the flavoenzyme glutathione reductase from rat liver. J Biol. Chem. 250:5474–5480.
13. Claiborne A (1985). Catalase activity. Ed: R. Greenwald, CRC Handbook of methods for oxygen radical research. CRC Press, Boca Raton, Fla, pp: 283-284.
14. Cohen E, Quistad GB, Casida JE (1996). Cytotoxicity of nimbolide, epoxyazadiradione and other limonoids from neem insecticide. Life Sci. 58: 1075–1081.
15. Cooper TG, Yeung CH, Nashan D, Nieschlag E (1988). Epididymal markers in human infertility. J Androl. 9:91– 101.
16. Devi J, Babu R (2023). Sharma Medicinal Importance of Azadirachta indica: An Overview. J Drug Delivery Therapeutics. 13(6):159-165.
17. Du Plessis SS, Agarwal A, Halabi J, Tvrda E (2015). Contemporary evidence on the physiological role of reactive oxygen species in human sperm function. J Assist Reprod Genet. 32: 509-520.
18. Glinsukon T, Somjaree R, Piyachaturawat P, Thebtaranonth Y (1986).Acute toxicity of nimbolide and nimbic acid in mice, rats and hamsters. Toxicol Lett. 30: 159–166.
19. Hammerstedt RH, Hay SR (1980). Effect of incubation temperature on motility and cAMP content of bovine sperm. Arch Biochem Biophys.199: 427–437.
20. He Z, Xie M, Li QQ, Duan J, Lu X (2022). Research Progress on the Microregulatory Mechanisms of Fertilization: A Review. In Vivo. 36(5): 2002–2013.
21. Holloway AJ, Moore HDM, Foster PMD (1990). The use of in vitro fertilization to detect reductions in the fertility of spermatozoa from males exposed to ethylene glycol monomethyl ether. Reprod Toxicol. 4:21-27.
22. Jin S, Yang W (2017). Factors and pathways involved in capacitation: how are they regulated? Oncotarget. 8:3600- 3627.
23. Kumar R, Mehta S, Pathak SR (2018). Bioactive constituents of neem. In: Synthesis of medicinal agents from plants. Elsevier, pp. 75- 103.
24. Kumbar SB, Jadaramkunti UC, Aladakatti RH (2012a). In vitro spermicidal efficacy of nimbolide, an isoprenoid of neem leaf, in albino rats. J Phytother Pharmacol. 1: 1-13.
25. Kumbar SB, Jadaramkunti UC, Aladakatti RH (2012b). In vitro effect of nimbolide, an isoprenoid of neem leaf, on antioxidant system of rat cauda epididymal spermatozoa: A dose dependent study. J Appl Pharmaceutical Sci. 2: 84-93.
26. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951). Protein measurement with folin phenol reagent. J Biol Chem. 193: 265–275.
27. M. H. Viljoen, M. S. Bornman, M. P. van der Merwe, D. J. du Plessis (2009). Alpha-glucosidase activity and sperm motility. Andrologia. 22(3):205-208.
28. Marklund S, Marklund G (1974). Involvement of superoxide anion radical in antioxidation of pyrogallol and a constituent assay for superoxide dismutase. Eur. J Biochem. 47:469–474.
29. Mohandas J, Marshall JJ, Duggin GG, Horvath JS, Tiller DJ (1984). Low activities of glutathione-related enzymes as factors in the genesis of urinary bladder cancer. Cancer. Res. 44: 5086–5091.
30. Nagini S, Nivetha R, Palrasu M, Mishra R (2021). Nimbolide, a Neem Limonoid, Is a Promising Candidate for the Anticancer Drug Arsenal. J Medicinal Chem. 64 (7): 3560-3577.
31. O’Flaherty C, Scarlata E (2022). Oxidative stress and reproductive function: The protection of mammalian spermatozoa against oxidative stress. Reproduction. 164: F67–F78.
32. Ohkawa H, Ohishi N, Yagi K (1979). Assay for lipid peroxidation in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95:351–358.
33. Pick E, Keisari Y (1981). Superoxide anion and H2O2 production by chemically elicited peritoneal macrophages-induced by multiple nonphagocytic stimuli. Cell. Immunol. 59:301–318.
34. Priyadarsini RV, Manikandan P, Kumar GH, Nagini S (2009). The neem limonoids azadirachtin and nimbolide inhibit hamster cheek pouch carcinogenesis by modulating xenobiotic-metabolizing enzymes, DNA damage, antioxidants, invasion and angiogenesis. Free Radical Research. 43(5):492-504.
35. Puga Molina LC, Luque GM, Balestrini PA, Marín Briggiler CI, Romarowski A, Buffone MG (2018). Molecular Basis of Human Sperm Capacitation. Front. Cell Dev. Biol. 6:Article 72(1-23).
36. Rodriguez-Miranda E, Buffone MG, Edwards EE, Ord TS, Lin K, Sammel MD, Gerton GL, Moss SB, William C (2008). Extracellular adenosine 5′-triphosphate alters motility and improves the fertilizing capability of mouse sperm. Biol Reprod. 79:164–171.
37. Schlingmann K, Michaut MA, McElwee JL, Wolff CA, Travis AJ, Turner RM (2007). Calmodulin and CaMKII in the sperm principal piece: evidence for a motility‑related calcium/ calmodulin pathway. J Androl. 28: 706–716.
38. Steegborn C (2014). Structure, mechanism, and regulation of soluble adenylyl cyclases‑similarities and differences to transmembrane adenylyl cyclases. Biochim Biophys Acta (BBA)-Mol Basis Dis. 1842: 2535–2547.
39. Suarez SS (2008). Control of hyperactivation in sperm. Hum Reprod Update. 14: 647–657.
40. Thompson A, Agarwal A, Du Plessis SS (2013). Physiological role of reactive oxygen species in sperm function: a review. In: Parekattil SJ, Agarwal A, edn. Antioxidants in male infertility: a guide for clinicians and researchers. New York, USA: Springer Science and Business Media, pp. 69–89.
41. Toyoda Y, Chang MC (1974a). Capacitation of epididymal spermatozoa in a medium with high K/Na ratio and cyclic AMP for the fertilization of rat eggs in vitro. J Reprod Fertil. 36:125-134.
42. Toyoda, Y and Chang, MC (1974b). Fertilization of rat eggs in vitro by epididymal spermatozoa: development of eggs following transfer. J Reprod Fertil. 36:9–22.
43. WHO (1999). Laboratory manual for the examination of human semen and sperm cervical mucus interaction. 4th ed. Cambridge: Cambridge University Press.
44. Woods J, Garside DA (1996). An in vivo and in vitro investigation into the effects of alpha- chlorohydrin on sperm motility and correlation with fertility in the Han Wistar rat. Reprod Toxicol. 103:199-207.
45. Yánez-Ortiz I, Catalán J, Delgado-Bermúdez A, Carluccio A, Miró J, Yeste M (2021). Addition of reduced glutathione (gsh) to freezing medium reduces intracellular ros levels in donkey sperm. Vet Sci. 8:302.