Maternal separation can affect the reproductive system by inflammasome activation in female mice


  • Kajal Khodamoradi Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
  • Hossein Amini-Khoei Medical Plants Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Sciences, Shahrekord, Iran
  • Zahra Khosravizadeh Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
  • Seyed Reza Hosseini Departent of Urology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
  • Ahmad Reza Dehpour Experimental Medicine Research Center, Tehran University of Medical Sciences, Tehran, Iran
  • Gholamreza Hassanzadeh Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran



Maternal separation stress, Inflammasome, Inflammation, Oxidative stress, Apoptosis, Reproductive system


Objective: The aim of this study is to investigate effect of maternal separation stress on the ovarian function in adult female mice.  

Methods: In this study, maternal separation in pups was performed during post-natal days (PND) 2 to 14. The histological alterations in ovarian tissue, ROS production (using DCFH-DA assay), gene expression of NLRP3, ASC, caspase-1, TLR4, BAX, BCL-2 and TNFα (using RT-PCR), protein levels of ATP, GPx, IL-1β and IL-18 (using ELISA). Also, protein expression of caspase-3 and NLRP3 (using immunocytochemistry) were evaluated.

Results: Results showed that maternal separation decreased percentage of primordial follicles while increased percentage of secondary and graafian follicles. In addition, maternal separation increased ROS production and decreased ATP and GPx concentrations. Furthermore, maternal separation significantly affected expression of cytokines and genes involved in inflammation and apoptosis including NLRP3, ASC, caspase-1, TLR4, TNFα, IL-1β, IL-18 BAX and BCL2. Findings also showed that stress-induced maternal separation significantly increased percentage of caspase-3 and NLRP3 positive cells. We concluded that maternal separation stress has harmful effects on ovarian tissue.

Conclusion: It seems that these harmful effects are probably occur through increase of ROS production and impact on mitochondrial function, inflammatory process and apoptosis pathways.


1. Tractenberg SG LM, de Azeredo LA, Orso R, Roithmann LG, Hoffmann ES, Brenhouse H, Grassi-Oliveira R. An overview of maternal separation effects on behavioural outcomes in mice: evidence from a four-stage methodological systematic review. Neurosci Biobehav Rev. 2016;68:489-503.
2. Harrison EL BB. Modulation of early stress-induced neurobiological changes: a review of behavioural and pharmacological interventions in animal models. Transl Psychiatry. 2014;4(5):e390.
3. Jóźków P MM. Psychological stress and the function of male gonads. Endokrynol Pol. 2012;63(1):44-9.
4. Ostanin AA AB, Aizikovich IV, Kozhin AY, Chernykh ER. Role of cytokines in the regulation of reproductive function. Bull Exp Biol Med. 2007;143(1):75-9.
5. M F. Stress-evoked sterile inflammation, danger associated molecular patterns (DAMPs), microbial associated molecular patterns (MAMPs) and the inflammasome. Brain Behav Immun. 2013;27:1-7.
6. Rock KL LE, Ontiveros F, Kono H. The sterile inflammatory response. Annu Rev Immunol. 2009;28:321-42.
7. Johnson JD CJ, Sharkey CM, Kennedy SL, Nickerson M, Greenwood BN, Fleshner M. Catecholamines mediate stress-induced increases in peripheral and central inflammatory cytokines. Neuroscience. 2005;35(4):1295-307.
8. Mazzeo RS DD, Fleshner M, Butterfield GE, Zamudio S, Wolfel EE, Moore LG. Interleukin-6 response to exercise and high-altitude exposure: influence of α-adrenergic blockade. J Appl Physiol. 2001;91(5):2143-9.
9. Frank MG TB, Watkins LR, Maier SF. Glucocorticoids mediate stress-induced priming of microglial pro-inflammatory responses. Brain Behav Immun. 2012;26(2):337-45.
10. Campisi J SC, Johnson JD, Asea A, Maslanik T, Bernstein-Hanley I, Fleshner M Stress-induced facilitation of host response to bacterial challenge in F344 rats is dependent on extracellular heat shock protein 72 and independent of alpha beta T cells. Stress. 2012;15(6):637-46.
11. Gómez López M DLA, Abarca Rojano E, Rojas Hernandez S, Martinez Godinez MD, Miliar Garcia A, Campos Rodriguez R. 17 β-Estradiol transcriptionally modulates Nlrp1 and Nlrp3 inflammasomes in gonadectomized rats with inflammation. Immunopharmacol Immunotoxicol. 2015;37(4):343-50.
12. Bazrafkan M, Nikmehr B, Shahverdi A, Hosseini SR, Hassani F, Poorhassan M, et al. Lipid Peroxidation and Its Role in the Expression of NLRP1a and NLRP3 Genes in Testicular Tissue of Male Rats: a Model of Spinal Cord Injury. Iran Biomed J. 2018;22(3):151-9.
13. Nikmehr B, Bazrafkan M, Hassanzadeh G, Shahverdi A, Sadighi Gilani MA, Kiani S, et al. The Correlation of Gene Expression of Inflammasome Indicators and Impaired Fertility in Rat Model of Spinal Cord Injury: A Time Course Study. Urology journal. 2017;14(6):5057-63.
14. Mohamadi Y, Noori Moghahi SMH, Mousavi M, Borhani-Haghighi M, Abolhassani F, Kashani IR, et al. Intrathecal transplantation of Wharton's jelly mesenchymal stem cells suppresses the NLRP1 inflammasome in the rat model of spinal cord injury. Journal of chemical neuroanatomy. 2019;97:1-8.
15. Yang CA CB. Inflammasomes and human autoimmunity: a comprehensive review. J Autoimmun. 2015;61:1-8.
16. Jin C FR. Molecular mechanism of NLRP3 inflammasome activation. J Clin Immunol. 2010;30(5):628-31.
17. Ghaffari N, Hassanzadeh G, Nowrouzi A, Gholaminejhad M, Mokhtari T, Seifali R, et al. Antioxidative and anti-inflammatory effects of Cichorium intybus L. seed extract in ischemia/reperfusion injury model of rat spinal cord. Journal of Contemporary Medical Sciences. 2018;4(4).
18. Shalini S DL, Dawar S, Kumar S Old, new and emerging functions of caspases. Cell death and differentiation. 2015 22(4):526.
19. Wagenmaker ER BK, Oakley AE, Tilbrook AJ, Karsch FJ. Psychosocial stress inhibits amplitude of gonadotropin-releasing hormone pulses independent of cortisol action on the type II glucocorticoid receptor. Endocrinology. 2009;150(2):762-9.
20. DJ K. Mitochondrial dysfunction, oxidative stress, regulation of exocytosis and their relevance to neurodegenerative diseases. J Neurochem. 2008 104(2):298-305.
21. RJ M. Mitochondrial antioxidants and the maintenance of cellular hydrogen peroxide levels. Oxid Med Cell Longev. 2018;2018.
22. Madrigal JL OR, Moro MA, Lizasoain I, Lorenzo P, Rodrigo J, Leza JC Glutathione depletion, lipid peroxidation and mitochondrial dysfunction are induced by chronic stress in rat brain. Neuropsychopharmacology. 2001;24(4):420.
23. Cecchino GN SE, da Motta EL, García-Velasco JA The role of mitochondrial activity in female fertility and assisted reproductive technologies: overview and current insights. Reprod Biomed Online. 2018;36(6):686-97.
24. Agarwal A A-MA, Premkumar BJ, Shaman A, Gupta S. The effects of oxidative stress on female reproduction: a review. Reprod Biol Endocrinol. 2012 10(1):49.
25. Amini-Khoei H AS, Shirzadian A, Haj-Mirzaian A, Alijanpour S, Rahimi-Balaei M, Mohammadi-Asl A, Hassanipour M, Mehr SE, Dehpour AR. Experiencing neonatal maternal separation increased the seizure threshold in adult male mice: involvement of the opioid system. Epilepsy Behav. 2015;52:37-41.
26. Amini-Khoei H AS, Mohammadi-Asl A, Alijanpour S, Poursaman S, Haj-Mirzaian A, Rastegar M, Mesdaghinia A, Banafshe HR, Sadeghi E, Samiei E. Experiencing neonatal maternal separation increased pain sensitivity in adult male mice: involvement of oxytocinergic system. Neuropeptides. 2017 61:77-85.
27. Amini-Khoei H H-SE, Beigi M, Soltani A, Mobini GR, Balali-Dehkordi S, Haj-Mirzaian A, Rafieian-Kopaei M, Alizadeh A, Hojjati MR, Validi M. On the role of corticosterone in behavioral disorders, microbiota composition alteration and neuroimmune response in adult male mice subjected to maternal separation stress. Int Immunopharmacol. 2019;66:242-50.
28. Dym M JM, Dirami G, Michael Price J, Rabin SJ, Mocchetti I, Ravindranath N. Expression of c-kit receptor and its autophosphorylation in immature rat type A spermatogonia. Biol Reprod. 1995;52(1):8-19.
29. Fatemi N SM, Shamsara M, Moayer F, Zavarehei MJ, Pouya A, Sayyahpour F, Ayat H, Gourabi H TBHP-induced oxidative stress alters microRNAs expression in mouse testis. J Assist Reprod Genet. 2014;31(10):1287-93.
30. Slotten HA KM, Hagan JJ, Marsden CA, Fone KC Long-lasting changes in behavioural and neuroendocrine indices in the rat following neonatal maternal separation: gender-dependent effects. Brain research. 2006 1097(1):123-32.
31. AS C. Social rearing effects on HPA axis activity over early development and in response to stress in rhesus monkeys. Developmental Psychobiology: The Journal of the International Society for Developmental Psychobiology. 1993;26(8):433-46.
32. Kuhn CM SS. Responses to maternal separation: mechanisms and mediators. Int J Dev Neurosci. 1998;16(3-4):261-70.
33. G M. Central nervous regulation of the hypothalamic-pituitary-adrenal axis and its impact on fertility, immunity, metabolism and animal welfare-a review. Archives Animal Breeding 2002;45(6):575-95.
34. Peng H ZW, Xiao T, Zhang Y Nlrp4g is an oocyte-specific gene but is not required for oocyte maturation in the mouse. Reproduction, Fertility and Development. 2014;26(5):758-68.
35. Tschopp J SK. NLRP3 inflammasome activation: The convergence of multiple signalling pathways on ROS production? Nat Rev Immunol. 2010 10(3):210.
36. Shimada K CT, Karlin J, Dagvadorj J, Chiba N, Chen S, Ramanujan VK, Wolf AJ, Vergnes L, Ojcius DM, Rentsendorj A. . Oxidized mitochondrial DNA activates the NLRP3 inflammasome during apoptosis. Immunity. 2012;36(3):401-14.
37. Shimada K CT, Karlin J, Chen S, Chiba N, Ramanujan VK, Vergnes L, Ojcius DM, Arditi M. Caspase-1 dependent IL-1b secretion is critical for host defense in a mouse model of Chlamydia pneumoniae lung infection. PLoS ONE. 2011;6(6):e21477.
38. Zhou R YA, Menu P, Tschopp J. A role for mitochondria in NLRP3 inflammasome activation. Nature. 2011 469(7329):221.
39. Nakahira K HJ, Rathinam VA, Lee SJ, Dolinay T, Lam HC, Englert JA, Rabinovitch M, Cernadas M, Kim HP, Fitzgerald KA. Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome. Nat Immunol. 2011 12(3):222.
40. Wree A MM, Inzaugarat ME, Eguchi A, Schuster S, Johnson CD, Peña CA, Geisler LJ, Papouchado BG, Hoffman HM, Feldstein AE. NLRP3 inflammasome driven liver injury and fibrosis: Roles of IL‐17 and TNF in mice. Hepatology. 2018;67(2):736-49.
41. McGeough MD WA, Inzaugarat ME, Haimovich A, Johnson CD, Peña CA, Goldbach-Mansky R, Broderick L, Feldstein AE, Hoffman HM TNF regulates transcription of NLRP3 inflammasome components and inflammatory molecules in cryopyrinopathies. J Clin Invest. 2017 127(12):4488-97.
42. Ighodaro OM AO. First line defence antioxidants-superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX): Their fundamental role in the entire antioxidant defence grid. Alexandria Journal of Medicine. 2018;54(4):287-93.
43. Agarwal A GS, Sekhon L, Shah R. Redox considerations in female reproductive function and assisted reproduction: from molecular mechanisms to health implications. Antioxidants & redox signaling. 2008;10(8):1375-404.
44. Agarwal A VG, Ong C, du Plessis SS Effect of oxidative stress on male reproduction. The world journal of men's health. 2014;32(1):1-7.
45. Lim J LU. Oxidative damage increases and antioxidant gene expression decreases with aging in the mouse ovary. Biology of reproduction. 2011;84(4):775-82.
46. Tatone C AF, Carbone MC, Monteleone P, Caserta D, Marci R, Artini PG, Piomboni P, Focarelli R. Cellular and molecular aspects of ovarian follicle ageing. Human reproduction update. 2008;14(2):131-42.
47. Behrman HR KP, Preston SL, Gao S Oxidative stress and the ovary. Journal of the Society for Gynecologic Investigation. 2001 8(1):40-2.
48. Jabbour H SK, Catalano R, Norman J Inflammatory pathways in female reproductive health and disease. Reproduction, Fertility and Development. 2009.
49. Herath S WE, Lilly ST, Gilbert RO, Dobson H, Bryant CE, Sheldon IM Ovarian follicular cells have innate immune capabilities that modulate their endocrine function. Reproduction, Fertility and Development. 2007;134(5):683-93.
50. Uri-Belapolsky S SA, Eliyahu E, Grossman H, Levi M, Chuderland D, Ninio-Many L, Hasky N, Shashar D, Almog T, Kandel-Kfir M. Interleukin-1 deficiency prolongs ovarian lifespan in mice. Proceedings of the National Academy of Sciences. 2014;111(34):12492-7.
51. MR H. Apoptosis in the ovary: molecular mechanisms. Human reproduction update. 2005;11(2):162-78.




How to Cite

Khodamoradi, K., Amini-Khoei, H., Khosravizadeh, Z., Hosseini, S. R., Dehpour, A. R., & Hassanzadeh, G. (2019). Maternal separation can affect the reproductive system by inflammasome activation in female mice. Journal of Contemporary Medical Sciences, 5(3), 125–130.

Most read articles by the same author(s)

1 2 > >>