Sertoli cell–conditioned medium restores spermatogenesis in azoospermic mouse testis

  • Sakineh Panahi
  • Amin Karamian
  • Ensieh Sajadi
  • Abbas Aliaghaei
  • Hamid Nazarian
  • Shabnam Abdi
  • Samira Danyali
  • Shahrokh Paktinat
  • Mohammad-Amin AbdollahifarEmail author
  • Reza Mastery FarahaniEmail author
Regular Article


The current study evaluates potential applications of Sertoli cell (SC)–conditioned medium (CM) and explores the effects of the conditioned medium on the spermatogenesis process in azoospermic mice. For this study, 40 adult mice (28–30 g) were divided into 4 experimental groups: (1) control, (2) DMSO 2% (10 μl), (3) busulfan (40 mg/kg single dose) and (4) busulfan/CM (10 μl). SCs were isolated from 4-week-old mouse testes. After using anesthetics, 10 μl of CM was injected over 3–5 min into each testis and subsequently, sperm samples were collected from the tail of the epididymis. Afterward, the animals were euthanized and testis samples were taken for histopathology experiments and RNA extraction in order to examine the expression of c-kit, STRA8 and PCNA genes. The data showed that CM notably increased the total sperm count and the number of testicular cells, such as spermatogonia, primary spermatocytes, round spermatids, SCs and Leydig cells compared with the control, DMSO and busulfan groups. Furthermore, the results showed that expression of c-kit and STRA8 was significantly decreased in the busulfan and busulfan/SC groups at 8 weeks after the last injection (p < 0.001) but no significant difference was found for PCNA compared with the control and DMSO groups (p < 0.05). These findings suggest that the Sertoli cell–conditioned medium may be beneficial as a practical approach for therapeutic strategies in reproductive and regenerative medicine.


Sertoli cell–conditioned medium Spermatogenesis Azoospermia 



This article has been extracted from the thesis by Mrs. Sakineh Panahi.

Author contributions

MAA and SP designed this study and conducted the stereological study and provided the clinical data and sample. AK and ES carried out the animal model and immunohistochemistry. AA and HN performed the statistical analysis. SA and SD wrote and drafted the manuscript. SP carried out the real-time PCR. VE is responsible for the English language and grammar editing. RMF helped in writing the draft of the manuscript and helped in designing this study. All authors read and approved the final manuscript.

Funding information

This study was financially supported by the Research Vice Chancellor of Shahid Beheshti University of Medical Sciences, Tehran, Iran (Registration No. 1395.115).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

The experimental protocol and all procedures performed in studies involving the use of animals were reviewed and approved by the Ethical Committee at Shahid Beheshti University of Medical Sciences (IR. SBMU. SM.REC.1396.184).


  1. Bellve A, Zheng W (1989) Growth factors as autocrine and paracrine modulators of male gonadal functions. J Reprod Fertil 85(2):771–793CrossRefGoogle Scholar
  2. Endo T, Romer KA, Anderson EL, Baltus AE, de Rooij DG, Page DC (2015) "Periodic retinoic acid–STRA8 signaling intersects with periodic germ-cell competencies to regulate spermatogenesis." Proc Natl Acad Sci: 201505683Google Scholar
  3. Ghaem Maghami R, Mirzapour T, Bayrami A (2018) Differentiation of mesenchymal stem cells to germ-like cells under induction of Sertoli cell-conditioned medium and retinoic acid. Andrologia 50(3):e12887CrossRefGoogle Scholar
  4. Grootenhuis A, Timmerman M, Hordijk P, de Jong F (1990) Inhibin in immature rat Sertoli cell conditioned medium: a 32 kDa αβ-B dimer. Mol Cell Endocrinol 70(1):109–116CrossRefGoogle Scholar
  5. Gundersen H, Bagger P, Bendtsen T, Evans S, Korbo L, Marcussen N, Møller A, Nielsen K, Nyengaard J, Pakkenberg B (1988a) The new stereological tools: disector, fractionator, nucleator and point sampled intercepts and their use in pathological research and diagnosis. Apmis 96(7–12):857–881CrossRefGoogle Scholar
  6. Gundersen H, BENDTSEN TF, KORBO L, MARCUSSEN N, Møller A, Nielsen K, Nyengaard J, Pakkenberg B, Sørensen FB, Vesterby A (1988b) Some new, simple and efficient stereological methods and their use in pathological research and diagnosis. Apmis 96(1–6):379–394CrossRefGoogle Scholar
  7. Hajian Monfared M, Akbari M, Solhjoo S, Tooli H, Omidi A, Aliakbari F, Ijaz S, Mokhtari T, Rastegar T (2017) Inductive role of sustentacular cells (Sertoli cells) conditioned medium on bone marrow derived mesenchymal stem cells. Int J Morphol 35(4):1597–1606CrossRefGoogle Scholar
  8. Hajihoseini M, Vahdati A, Ebrahim Hosseini S, Mehrabani D, Tamadon A (2017) Induction of spermatogenesis after stem cell therapy of azoospermic guinea pigs. Vet Arh 87(3):333–350Google Scholar
  9. Jarvi K, Lo K, Fischer A, Grantmyre J, Zini A, Chow V, Mak V (2010) CUA Guideline: The workup of azoospermic males. Can Urol Assoc J 4(3):163Google Scholar
  10. Ma H-T, Niu C-M, Xia J, Shen X-Y, Xia M-M, Hu Y-Q, Zheng Y (2018) Stimulated by retinoic acid gene 8 (Stra8) plays important roles in many stages of spermatogenesis. Asian J Androl 20(5):479Google Scholar
  11. Medrano J, Marques-Mari A, Aguilar C, Riboldi M, Garrido N, Martinez-Romero A, O'connor E, Gil-Salom M, Simon C (2010) Comparative analysis of the germ cell markers c-KIT, SSEA-1 and VASA in testicular biopsies from secretory and obstructive azoospermias. Mol Hum Reprod 16(11):811–817Google Scholar
  12. Monfared MH, Minaee B, Rastegar T, Khrazinejad E, Barbarestani M (2016) Sertoli cell condition medium can induce germ like cells from bone marrow derived mesenchymal stem cells. Iran J Basic Med Sci 19(11):1186Google Scholar
  13. Monsefi M, Fereydouni B, Rohani L, Talaei T (2013) Mesenchymal stem cells repair germinal cells of seminiferous tubules of sterile rats. Iran J Reprod Med 11(7):537Google Scholar
  14. Mozafar A, Mehrabani D, Vahdati A, Hosseini E, Forouzanfar M (2018) Histomorphometric evaluation of allogeneic transplantation of bone marrow mesenchymal stem cells in azoospermic mice model. lnaBJ 10(2):171–178Google Scholar
  15. Nayernia K, Lee JH, Drusenheimer N, Nolte J, Wulf G, Dressel R, Gromoll J, Engel W (2006) Derivation of male germ cells from bone marrow stem cells. Lab Investig 86(7):654CrossRefGoogle Scholar
  16. Niedenberger BA, Busada JT, Geyer CB (2015) Marker expression reveals heterogeneity of spermatogonia in the neonatal mouse testis. Reproduction 149(4):329–338CrossRefGoogle Scholar
  17. Perrard-Sapori M, Chatelain P, Rogemond N, Saez J (1987) Modulation of Leydig cell functions by culture with Sertoli cells or with Sertoli cell-conditioned medium: effect of insulin, somatomedin-C and FSH. Mol Cell Endocrinol 50(3):193–201CrossRefGoogle Scholar
  18. Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30(9):e36–e36CrossRefGoogle Scholar
  19. Rebourcet D, Wu J, Cruickshanks L, Smith SE, Milne L, Fernando A, Wallace RJ, Gray CD, Hadoke PW, Mitchell RT (2016) Sertoli cells modulate testicular vascular network development, structure, and function to influence circulating testosterone concentrations in adult male mice. Endocrinology 157(6):2479–2488CrossRefGoogle Scholar
  20. Rossi P, Dolci S, Sette C, Capolunghi F, Pellegrini M, Loiarro M, Di Agostino S, Paronetto MP, Grimaldi P, Merico D (2004) Analysis of the gene expression profile of mouse male meiotic germ cells. Gene Expr Patterns 4(3):267–281CrossRefGoogle Scholar
  21. Takashima S, Kanatsu-Shinohara M, Tanaka T, Morimoto H, Inoue K, Ogonuki N, Jijiwa M, Takahashi M, Ogura A, Shinohara T (2015) Functional differences between GDNF-dependent and FGF2-dependent mouse spermatogonial stem cell self-renewal. Stem Cell Reports 4(3):489–502Google Scholar
  22. Zanganeh BM, Rastegar T, Roudkenar MH, Kashani IR, Amidi F, Barbarestani M (2013) Co-culture of spermatogonial stem cells with sertoli cells in the presence of testosterone and FSH improved differentiation via up-regulation of post meiotic genes. Acta Med Iran 51(1):1–11Google Scholar
  23. Zhang L, Tang J, Haines CJ, Feng H, Lai L, Teng X, Han Y (2011) c-kit and its related genes in spermatogonial differentiation. Spermatogenesis 1(3):186–194CrossRefGoogle Scholar
  24. Zhang L, Tang J, Haines CJ, Feng H, Lai L, Teng X, Han Y (2013) c-kit expression profile and regulatory factors during spermatogonial stem cell differentiation. BMC Dev Biol 13(1):38CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Biology and Anatomical Sciences, School of MedicineShahid Beheshti University of Medical SciencesTehranIran
  2. 2.Department of Anatomical Sciences & Cognitive Neuroscience, Faculty of Medicine, Tehran Medical SciencesIslamic Azad UniversityTehranIran
  3. 3.Department of Physiology, School of MedicineShahid Beheshti University of Medical SciencesTehranIran

Personalised recommendations