Ultrastructural and histological study of testicular torsion-detorsion on rat and protective effects of fibroblast and macrophage conditioned medium

Document Type : Original Article

Authors

1 Department of Basic Sciences, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran

2 Department of Anatomy, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran

3 Department of Surgery and Diagnostic Imaging; Faculty of Veterinary Medicine, Urmia University, Urmia, Iran

4 Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Abstract
Testicular torsion-detorsion (TD) or ischemia/reperfusion causes reactive oxygen species over-production and has extensive destructive effects on testicular tissue. Following TD, this study evaluated the therapeutic effects of conditioned medium (CM) of fibroblast, macrophage, and co-culture on testicular histomorphometric and ultrastructure. Adult rats were divided into 7 groups. Healthy control, Control sham, TD, and Dulbecco's Modified Eagle Medium (DMEM) groups. Experimental groups: fibroblast, macrophage, and co-culture. 10µl supernatant was obtained from the separate cultures of fibroblasts, and macrophages and co-culture were injected. All injections were made through rete testis. 35 days after the operation, the testis was sampled for histomorphometric and ultrastructural studies. However, the ultrastructural study of testicular tissue also showed that extensive changes occurred in Sertoli cells' nucleus, nucleolus, and mitochondria. This study showed that in fibroblast and slightly less in macrophage groups, there were good improvements in all histomorphometric and ultrastructure parameters similar to the healthy control group. It was also shown that the DMEM group had slightly better recovery conditions than the TD group, but the co-culture group showed similar conditions to the TD. Overall, it can be concluded that the CM of fibroblast was very effective. In the groups receiving fibroblastic and macrophage CM, all parameters exhibited favorable improvement but in the fibroblast group, most of the parameters were similar to those in the healthy controls group. It was also shown that the CM of fibroblast-macrophage co-culture could not be improved on TD condition but made it worsened.

Keywords

Subjects

1.     Afroughi S, Pouzesh M. Prevalence and risk factors of infertility in a sample of Iranian couples. J Biom Biostat 2018; 9(4): 407. doi: 10.4172/2155-6180.1000407.
2.     Cocuzza M, Alvarenga C, Pagani R. The epidemiology and etiology of azoospermia. Clinics (Sao Paulo) 2013; 68(Suppl 1): 15-26.
3.     Samy A, El-Adl M, Rezk S, et al. The potential protective and therapeutic effects of platelet-rich plasma on ischemia/reperfusion injury following experimental torsion/detorsion of testis in the Albino rat model. Life Sci 2020; 256: 117982. doi: 10.1016/j.lfs. 2020.117982. 
4.     Khan SA, Khan SJ, Dorrington JH. Interleukin-1 stimulates deoxyribonucleic acid synthesis in immature rat Leydig cells in vitro. Endocrinology 1992; 131(4): 1853-1857.
5.     Elshaari FA, Elfagih RI, Sheriff DS, et al. Testicular torsion-detorsion-histological and biochemical changes in rat testis. J Cytol Histol 2012; 3(1): 1000316. doi: 10.4172/2157-7099.1000136.
6.     Kabay S, Ozden H, Guven G, et al. Protective effects of the nuclear factor kappa B inhibitor pyrrolidine dithiocarbamate on experimental testicular torsion and detorsion injury. Korean J Physiol Pharmacol 2014; 18(4): 321-326.
7.     Inan M, Basaran UN, Dokmeci D, et al. Methylene blue increases contralateral testicular ischaemia–reperfusion injury after unilateral testicular torsion. Clin Exp Pharmacol Physiol 2008; 35(1): 50-54.
8.     Yun YR, Won JE, Jeon E, et al. Fibroblast growth factors: biology, function, and application for tissue regeneration. J Tissue Eng 2010; 2010: 218142. doi: 10.4061/2010/218142.
9.     Zhang X, Goncalves R, Mosser DM. The isolation and characterization of murine macrophages. Curr Protoc Immunol 2008; Chapter 14: 14.1.1-14.1.14. doi: 10.1002/0471142735.im1401s83.
10. DeFalco T, Potter SJ, Williams AV, et al. Macrophages contribute to the spermatogonial niche in the adult testis. Cell Rep 2015; 12(7): 1107-1119.
11. Paré B, Deschênes LT, Pouliot R, et al. An optimized approach to recover secreted proteins from fibroblast conditioned-media for secretomic analysis. Front Cell Neurosci 2016; 10: 70. doi: 10.3389/fncel.2016.00070.
12. Kim HO, Choi SM, Kim HS. Mesenchymal stem cell-derived secretome and microvesicles as a cell-free therapeutics for neurodegenerative disorders. Tissue Eng Regen Med 2013; 10: 93-101.
13. Cantinieaux D, Quertainmont R, Blacher S, et al. Conditioned medium from bone marrow-derived mesenchymal stem cells improves recovery after spinal cord injury in rats: an original strategy to avoid cell transplantation. PloS One 2013; 8(8): e69515. doi: 10.1371/journal.pone.0069515. 
14. Ivanova-Todorova E, Bochev I, Dimitrov R, et al. Conditioned medium from adipose tissue‐derived mesenchymal stem cells induces CD4+ FOXP3+ cells and increases IL‐10 secretion. J Biomed Biotechnol 2012; 2012: 295167. doi: 10.1155/2012/295167.
15. Pawitan JA. Prospect of stem cell conditioned medium in regenerative medicine. Biomed Res Int 2014; 2014: 965849. doi: 10.1155/2014/965849.
16. Chou YS, Young TH, Lou PJ. Effects of biomaterial-derived fibroblast conditioned medium on the α- amylase expression of parotid gland acinar cells. Acta Biomater 2015; 27: 214-223.
17. Hur W, Lee HY, Min HS, et al. Regeneration of full-thickness skin defects by differentiated adipose-derived stem cells into fibroblast-like cells by fibroblast-conditioned medium. Stem Cell Res Ther 2017; 8(1): 92. doi: 10.1186/s13287-017-0520-7.
18.     Amirjamshidi H, Milani BY, Sagha HM, et al. Limbal fibroblast conditioned media: a non-invasive treatment for limbal stem cell deficiency. Mol Vis 2011; 17:658-666.
19. Burguera EF, Bitar M, Bruinink A. Novel in vitro co-culture methodology to investigate heterotypic cell-cell interactions. Eur Cell Mater 2010; 19: 166-179.
20. Bogdanowicz DR, Lu HH. Multifunction co-culture model for evaluating cell-cell interactions. Methods Mol Biol. 2014; 1202: 29-36.
21. Mechlin CW, Levesque J, Feustel P, et al. Mast cell numbers negatively correlate with fibrosis in cryptorchid testes. J Pediatr Urol 2014; 10(3): 527-531.
22. Adelodun ST, Adewole OS, Bejide RA, et al. Protective effects of Vitex doniana (black plum) against ischemic testes torsion injury: histological and morphometric features. Pathophysiology 2016; 23(3): 157-168.
23. Mossadegh-Keller N, Gentek R, Gimenez G, et al. Developmental origin and maintenance of distinct testicular macrophage populations. J Exp Med2017; 214(10): 2829-2841.
24. Calkins JH, Sigel MM, Nankin HR, et al. Interleukin-1 inhibits Leydig cell steroidogenesis in primary culture. Endocrinology 1988; 123(3): 1605-1610.
25. Verhoeven G, Cailleau J, Van Damme J, et al. Interleukin-1 stimulates steroidogenesis in cultured rat Leydig cells. Mol Cell Endocrinol 1988; 57(1-2): 51-60.
26. Holt DJ, Chamberlain LM, Grainger DW. Cell-cell signaling in co-cultures of macrophages and fibroblasts. Biomaterials 2010; 31(36): 9382-9394.
27. Abo‐Elmaksoud A, Sinowatz F. Expression and localization of growth factors and their receptors in the mammalian testis. Part I: Fibroblast growth factors and insulin‐like growth factors. Anat Histol Embryol 2005; 34(5): 319-334.
28. Lai MS, Wang CY, Yang SH, et al. The expression profiles of fibroblast growth factor 9 and its receptors in developing mice testes. Organogenesis 2016; 12(2): 61-77.
29. Mirzaei M, Shahrooz R, Shalizar Jalali A, et al. The effects of fibroblast and macrophage separated and co-culture conditioned medium on sperm parameters disorders induced by testicular torsion-detorsion in adult rats [Persian]. Fasa Univ Med Sci 2019; 9(3): 1564-1576.
30. Kim Y, Bingham N, Sekido R, et al. Fibroblast growth factor receptor 2 regulates proliferation and Sertoli differentiation during male sex determination. Proc Natl Acad Sci U S A 2007; 104(42): 16558-16563.
31. Hafez MS, Makhlouf NA, Saleh HA. The effect of detorsion versus orchiectomy following experimental unilateral testicular torsion on the contralateral testis and epididymis: a light and transmission electron microscopic study. Egypt J Histol 2012; 35(3): 620-632.
32.     Amiri M, Roghani M, Aminzadeh M, et al. The effect of fibroblast growth factor 21 on a cellular model of Alzheimer's disease with emphasis on cell viability and mitochondrial membrane potential. JBCP 2017; 5(2): 41-46.
Volume 17, Issue 5
May 2026
Pages 361-369

  • Receive Date 01 March 2025
  • Revise Date 22 August 2025
  • Accept Date 11 October 2025