The Effect of Stem Cell Factor and Retinoids in Spermatogonial Cell Differentiation of Testicular Tissue of Obstructive Azoospermia Model Adult Rats

Subject Areas : Journal of Animal Biology

Mahnaz Nasimi ¹ , Esmaeil Fattahi ² , Sayed Gholamali Jorsaraei ³ , Maryam Gholamitabar Tabari ⁴ , Ebrahim Zabihi Neyshabouri ⁵

1 - Department of Biology, Ayatollah Amoli Branch, Islamic Azad University, Amol, Iran
2 - گروه زیست شناسی، واحد آیت ا... آملی، دانشگاه آزاد اسلامی، آمل، ایران
3 - 2- Department of Anatomical Sciences, Infertility and Reproductive Health Research Center, Research Institute for Health, Babol University of Medical Sciences, Babol, Iran
4 - Department of Midwifery, Research center for Fertility Health, Sari Branch, Islamic Azad University, Sari, Iran
5 - Department of Pharmacology, Cellular and Molecular Biology Research Center, Health Research Institute, Babol University of Medical Sciences, Babol, Iran

Received: 2019-08-16 Accepted : 2020-02-12 Published : 2020-02-20

Keywords: tissue culture, Rat, Retinoids, Stem Cell Factor, Obstructive Azoospermia,

Abstract :

Retinoids and stem cell factor play an important role in the stem cells proliferation, survival, and differentiation into sperm. Therefore, in this study, the effect of stem cell factor and retinoids on the proliferation and differentiation of spermatogonial cells in testis seminiferous tubules of obstructive azoospermia model adult rats was investigated using the tissue culture system. The testicular tissue fragments were cultured in the culture medium supplement with retinoids or stem cell factor, or a combination of both at the temperature of 34.5 °C and 5% CO2 for 25, 30, and 35 days. Then, the stained tissue sections prepared at the mentioned times were evaluated using PAS and hematoxylin staining methods. The cell counting per unit area was done using ImageJ software, and the statistical analysis was performed by one-way ANOVA and Tukey's post hoc test. The number of spermatogonia, spermatocyte, and Leydig cells increased significantly on day 25, 30 and 35 of culture; additionally, the number of round and elongated spermatid increased significantly on day 35 of culture in the testicular tissue of the obstructive azoospermia compared to the experimental and control groups (p < 0.001). Histological examination of testicular tissue in different culture conditions revealed pathological changes in the epithelialmaking cells. Retinoids played a more effective role on day 35 of culture compared to other factors in maintaining and proliferation of cell lines of the spermatogenesis process. As these factors are not alone the cause of proliferation and differentiation induction, therefore optimizing the culture medium is recommended to achieve adult sperm.

References:

1. Allard E.K., Boekelheide K., 1996. Fate of germ cells in 2,5-hexanedioneinduced testicular injury. II. Atrophy persists due to a reduced stem cell mass and ongoing apoptosis. Toxicol Appl Pharmacol, 137: 149-156.

2. Arkoun B., Dumont L., Milazzo J.P., Way A., Bironneau A., Wils J., Mace B., Rives N., 2015. Retinol improves in vitro differentiation of pre-pubertal mouse spermatogonial stem cells into sperm during the first wave of spermatogenesis. Plos One, 10(2): e0116660.

3. Baker K., Sabanegh Jr E., 2013. Obstructive azoospermia: reconstructive techniques and results. Clinics, 68(S1): 61-73.

4. Bergh A., Collin O., Lissbrant E., 2001. Effects of acute graded reductions in testicular blood flow on testicular morphology in the adult rat. Biology of reproduction, 64(1): 13–20.

5. Bokemeyer C., Kuczyk M.A., Dunn T., Serth J., Hartmann K., Jonasson J., Pietsch T., Jonas U.,  Schmoll H.J., 1996. Expression of stem-cell factor and its receptor c-kit protein in normal testicular tissue and malignant germ-cell tumours. J Cancer Res Clin Oncol, 122(5): 301-306.

6. Dumont L., Arkoun B., Jumeau F., Milazzo J.P., Bironneau A., Liot D., Wils J., Rondanino C., Rives N., 2015. Assessment of the optimal vitrification protocol for pre-pubertal mice testes leading to successful in vitro production of flagellated spermatozoa. Andrology, 3: 611–625.

7. Duru F.I.O., Ajayi S., Azu O.O., 2013. The effect of unilateral vasectomy on testosterone and testicular parameters in the adult male African giant rat (Cricetomys Gambianus). African Health Sciences, 13(2): 483 - 489.

8. Farias J.G., Bustos-Obregon E., Orellana R., Bucarey J.L., Quiroz E., Reyes J.G., 2005. Effects of chronic hypobaric hypoxia on testis histology and round spermatid oxidative metabolism. Andrologia, 37(1): 47–52.

9. Ghieh F., Mitchell V., Mandon-Pepin B and Vialard F., 2019. Genetic defects in human azoospermia. Basic and Clinical Andrology, 29: 4.

10. Gholamitabar Tabari M., Jorsaraei S.G.A., Ghasemzadeh-Hasankolaei M., Ahmadi A.A., Amirikia M., 2018. Evaluation of Novel Mouse-Specific Germ Cell Gene Expression in Embryonic Stem Cell-Derived Germ Cell-Like Cells In Vitro with Retinoic Acid Treatment. Cellular reprogramming, 20(4): 245-255.

11. Gohbara A., Katagiri K., Sato T., Kubota Y., Kagechika H., Araki Y., et al., 2010. In vitro murine spermatogenesis in an organ culture system. Biology of Reproduction, 83: 261-267.

12. Grimaldi P., Rossi P., Dolci S., Ripamonti C.B., Geremia R., 2002. Molecular genetics of male infertility: stem cell factor/c-kit system. Am J Reprod Immunol, 48(1): 27-33.

13. Hai Y., Hou J., Liu Y., Liu Y., Yang H., Li Z., He Z., 2014. The roles and regulation of Sertoli cells in fate determinations of spermatogonial stem cells and spermatogenesis. Semin Cell Dev Biol, 29: 66-75.

14. Hasanzadeh S., Najafi G., Pirdehghan H.R., Bonyadi F., 2016. The Effects of Ceftriaxone on Histology, Histomorphometry, and Histochemistry of Testis and Sperm Characteristics in Mice. Qom Univ Med Sci J, 10(6) :1-12.

15.Hogarth C.A., Griswold M.D., 2013. Retinoic acid regulation of male meiosis, Current Opinion in Endocrinology, Diabetes, and Obesity, 20(3): 217-223.

16. Kim K.J., Kim B.G., Kim Y.H., Lee Y.A., Kim B.J., Jung S.E., et al., 2015. In Vitro Spermatogenesis Using Bovine Testis Tissue Culture Techniques. Tissue Engineering and Regenerative Medicine, 12(5): 314-323.

17. Koruji S.M., Movahedin M., Mowla S.J., Gorabi H., Jabari Arfaei A., 2007. The Effects of Inducer Factors on Adult Mouse Spermatogonial Cells Colony Formation In Vitro. Yakhteh Medical Journal,9(2): 141-150.

18. Kovac J.R., Lehmann K.J., Fischer M.A., 2014. A single-center study examining the outcomes of percutaneous epididymal sperm aspiration in the treatment of obstructive azoospermia. Urol Ann, 6: 41–45.

19. Kubota H., Avarbock M.R., Brinster R.L., 2004. Culture conditions and single growth factors affect fate determination of mouse spermatogonial stem cells. Biol Reprod, 71: 722-731.

20. Lavers Ann E., Swanlund David J., Hunter Brian A., Tran Michael L., Pryor Jon L., Roberts K.P., 2006. Acute Effect of Vasectomy on the Function of the Rat Epididymal Epithelium and Vas Deferens. Andrology, 27: 326-336.

21. Liu F., Cai C., Wu X., Cheng Y., Lin T., Wei G., He D., 2016. Effect of knockOut serum replacement on germ cell development of immature testis tissue culture. Theriogenology, 85: 193–199.

22. Martin D., 1994. Basement Membrane Regulation of Sertoli Cells. Endocrinol  Rev, 15: 102–115.

23. Park T.S., Han J.Y., 2000. Derivation and characterization of pluripotent embryonic germ cells in chicken. Mol Reprod Dev, 56: 475-482.

24. Pierce G.B., Nakane P.K., 1969. Basement membranes. Synthesis and deposition in response to cellular injury. Lab Invest, 21(1): 27-41.

25. Poongothai J., Gopenath T.S., Manonayaki S., 2009. Genetics of human male infertility. Singapore Med J, 50(4): 336-347.

26. Qin Q., Liu J., Ma Y., Wang Y.,Zhang F., Gao S., Dong L., 2017. Aberrant expressions of stem cell factor/c-KIT in rat testis with varicocele. Journal of the Formosan Medical Association, 116: 542-548.

27.Reda A., Hou M., Landreh L., Kjartansdottir K.R., Svechnikov K., Soder O., Stukenborg J.B., 2014. In vitro spermatogenesis – optimal culture conditions for testicular cell survival, germ cell differentiation, and steroidogenesis in rats. Front Endocrinol, 5: 21.

28. Reda A., Hou M., Winton T.R., Chapin R.E., Soder O., Stukenborg J.B., 2016. In vitro differentiation of rat spermatogonia into round spermatids in tissue culture. Molecular Human Reproduction, 22(9): 601–612.

29. Russell L.D., Ettlin R.A., Sinha Hikim A.P., Clegg E.D., 2008. Histological and histopathological evaluation of the testis. International Journal of Andrology, 16(1): 83 - 83.

30. Salomon F., Saremaslani P., Jakob M., Hedinger C.E., 1982. Immune complex orchitis in infertile men. Immunoelectron microscopy of abnormal basement membrane structures. Technical Methods and Pathology, 47(6): 555-567.

31. Sato T., Katagiri K., Gohbara A., Inoue K., Ogonuki N., Ogura A., et al., 2011a. In vitro production of functional sperm in cultured neonatal mouse testes. Nature, 471: 504-507.

32. Sato T.,   Katagiri K.,   Kojima K.,  Komeya M.,   Yao M.,  and Ogawa T., 2015. In Vitro Spermatogenesis in Explanted Adult Mouse Testis Tissues. PLoS One, 10(6): e0130171.

33. Schlegel P.N., 2004. Cause of azoospermia and their management. Reproduction, Fertility and Development Journal, 16: 561-572.

34. Skinner M.K., Tung P.S., Fritz I.B.,1985. Cooperativity between Sertoli cells and testicular peritubular cells in the production and deposition of extracellular matrix components. J Cell Biol, 100(6): 1941-1947.

35. Steinberger A., Steinberger E., Perloff W.H., 1964. Mammalian Testes in Organ Culture. Experimental Cell Research, 36: 19-27.

36. Steinberger A., Article: M.J., 1975. In vitro techniques for the study of spermatogenesis. Enzymology Journal Article, 39: 283-296.

37. Steinberger E., Steinberger A., Perloff W.H., 1964. Initiation of spermatogenesis in vitro. Endocrinology, 74: 788–792.

38. Travers A., Arkoun B., Safsaf A., Milazzo J.P., Absyte A., et al., 2013. Effects of vitamin A on In Vitro maturation of pre-pubertal mouse spermatogonial stem cells. Plos One, 8(12): e82819.

39.Vernet N., Dennefeld C., Klopfenstein M., et al., 2008. RXR beta expression in Sertoli cells controls cholesterol homeostasis and spermiation. Reproduction,136: 619-626.

40. Wang S., Wang X., Ma L., Lin X., Zhang D., et al., 2016. Retinoic Acid Is Sufficient for the In Vitro Induction of Mouse Spermatocytes. Stem Cell Reports j, 7: 80–94.

41. Yan W., Suominen J and Toppari J., 2000. Stem cell factor protects germ cells from apoptosis in vitro. Journal of Cell Science, 113: 161-168.

42.Yokonishi T., Sato T., Katagiri K., and Ogawa T., 2013. In Vitro Spermatogenesis Using an Organ Culture Technique. Methods in molecular biology, 927: 479-488.

43. Zhang L., Tang J., Haines C.J., Feng H., Teng X., Han Y., 2015. RA induces differentiation of multipotent P19 cells towards male germ cell. In Vitro Cellular and  Developmental Biology- Animals, 51: 85-91.