Animal Reproduction (AR)
https://animal-reproduction.org/article/doi/10.1590/1984-3143-AR2020-0257
Animal Reproduction (AR)
ORIGINAL ARTICLE

The role of Rho-associated kinase inhibitor, Y-27632 on primary culture of ovine spermatogonial stem cells

Fatemeh Emamdoust; Mehdi Aminafshar; Mohammad Zandi; Mohammad Reza Sanjabi

Downloads: 0
Views: 30

Abstract

The access to sufficient numbers of spermatogonial stem cells (SSCs) is a prerequisite for the study of their regulation and further biomanipulation. Rho kinase (ROCK) belongs to a family of serine/threonine kinases and involves in a wide range of fundamental cellular functions. The aim of the present study was to study the effect of ROCK inhibitor, Y-27632 (0.1-40 µM), during the primary culture of ovine SSCs. SSCs were collected from 3-5-month-old’s lamb testes. The viability of SSCs, the apoptosis assay of SSCs, the intracellular reactive oxygen species (ROS) analysis, and the SSCs markers and apoptosis-related gene expressions were detected by MTT reduction assay, Annexin V–FITC/ Propidium Iodide (PI) dual staining, flow cytometry and real-time-PCR studies, respectively. Morphological analyses indicated that the 5-10 µM Y-27632 had an optimal effect on the number of presumptive SSCs colonies and the area covered by them after a 10 days culture. The cell viability, apoptosis and necrosis of SSCs after 10 days’ culture were not affected in comparison with the control group, and the 20 µM of Y-27632 resulted in significantly decreased cell viability (P<0.05) and an increased necrosis of cells. On day 10 after culture, the expression of P53 was decreased with an increase from 0 to 10 µM in the Y-27632 dose. In the 20 µM Y-27632 group, the expressions of P53 and Bax were higher and the Bcl-2 was lower than other groups and these values were significantly different from 5 and 10 µM Y-27632 groups (P<0.05). The level of intracellular ROS was decreased with an increase in the Y-27632 dose from 5 to 20 µM in comparison with the control group. In conclusion, the present study demonstrated that Y-27632 at a concentration of 5-10 µM provided optimal culture conditions for the primary culture of ovine SSCs.

Keywords

spermatogonial stem cells, primary culture, Y-27632, sheep

References

Abbasi H, Tahmoorespur M, Hosseini SM, Nasiri Z, Bahadorani M, Hajian M, Nasiri MR, Nasr-Esfahani MH. THY1 as a reliable marker for enrichment of undifferentiated spermatogonia in the goat. Theriogenology. 2013;80(8):923-32. http://dx.doi.org/10.1016/j.theriogenology.2013.07.020. PMid:23987985.

Azizi H, Hamidabadi HG, Skutella T. Differential proliferation effects after short-term cultivation of mouse spermatogonial stem cells on different feeder layers. Cell J. 2019;21(2):186-93. PMid:30825292.

Bi YL, Zhou Q, Du F, Wu MF, Xu GT, Sui GQ. Regulation of functional corneal endothelial cells isolated from sphere colonies by Rho-associated Protein kinase inhibitor. Exp Ther Med. 2013;5(2):433-7. http://dx.doi.org/10.3892/etm.2012.816. PMid:23408756.

Binsila KB, Selvaraju S, Ghosh SK, Parthipan S, Archana SS, Arangasamy A, Prasad JK, Bhatta R, Ravindra JP. Isolation and enrichment of putative spermatogonial stem cells from ram (Ovis aries) testis. Anim Reprod Sci. 2018;196:9-18. http://dx.doi.org/10.1016/j.anireprosci.2018.04.070. PMid:29861343.

Borjigin U, Davey R, Hutton K, Herrid M. Expression of promyelocytic leukemia zinc-finger in ovine testis and its application in evaluating the enrichment efficiency of differential plating. Reprod Fertil Dev. 2010;22(5):733-42. http://dx.doi.org/10.1071/RD09237. PMid:20450825.

Bosseboeuf A, Gautier A, Auvray P, Mazan S, Sourdaine P. Characterization of spermatogonial markers in the mature testis of the dogfish (Scyliorhinus canicula L.). Reproduction. 2013;147(1):125-39. http://dx.doi.org/10.1530/REP-13-0316. PMid:24123129.

Cory S, Adams JM. The Bcl2 family: regulators of the cellular lifeor-death switch. Nat Rev Cancer. 2002;2(9):647-56. http://dx.doi.org/10.1038/nrc883. PMid:12209154.

Costoya JA, Hobbs RM, Barna M, Cattoretti G, Manova K, Sukhwani M, Orwig KE, Wolgemuth DJ, Pandolfi PP. Essential role of Plzf in maintenance of spermatogonial stem cells. Nat Genet. 2004;36(6):653-9. http://dx.doi.org/10.1038/ng1367. PMid:15156143.

Dobrinski I. Transplantation of germ cells and testis tissue to study mammalian spermatogenesis. Anim Reprod. 2006;3:135-45.

Echeverry N, Bachmann D, Ke F, Strasser A, Simon HU, Kaufmann T. Intracellular localization of the BCL-2 family member BOK and functional implications. Cell Death Differ. 2013;20(6):785-99. http://dx.doi.org/10.1038/cdd.2013.10. PMid:23429263.

Emre N, Vidal JG, Elia J, O’Connor ED, Paramban RI, Hefferan MP, Navarro R, Goldberg DS, Varki NM, Marsala M, Carson CT. The ROCK Inhibitor Y-27632 improves recovery of human embryonic stem cells after fluorescence activated cell sorting with multiple cell surface markers. PLoS One. 2010;5(8):e12148. http://dx.doi.org/10.1371/journal.pone.0012148. PMid:20730054.

Heidari B, Rahmati-Ahmadabadi M, Akhondi MM, Zarnani AH, Jeddi-Tehrani M, Shirazi A, Naderi MM, Behzadi B. Isolation, identification, and culture of goat spermatogonial stem cells using c-kit and PGP9.5 markers. J Assist Reprod Genet. 2012;29(10):1029-38. http://dx.doi.org/10.1007/s10815-012-9828-5. PMid:22782689.

Heng BC. Effect of Rho-associated kinase (ROCK) inhibitor Y-27632 on the post-thaw viability of cryopreserved human bone marrow-derived mesenchymal stem cells. Tissue Cell. 2009;41(5):376-80. http://dx.doi.org/10.1016/j.tice.2009.01.004. PMid:19261317.

Hong N, Li Z, Hong Y. Fish stem cell cultures. Int J Biol Sci. 2011;7(4):392-402. http://dx.doi.org/10.7150/ijbs.7.392. PMid:21547056.

Hwang IS, Hara H, Chung HJ, Hirabayashi M, Hochi S. Rescue of vitrified-warmed bovine oocytes with Rho-associated coiled-coil kinase inhibitor1. Biol Reprod. 2013;89(2):26. http://dx.doi.org/10.1095/biolreprod.113.109769. PMid:23782835.

Izadyar F, Matthijs-Rijsenbilt JJ, Ouden K, Creemers LB, Woelders H, Rooij DG. Development of a cryopreservation protocol for type A spermatogonia. J Androl. 2002;23(4):537-45. PMid:12065461.

Kala S, Kaushik R, Singh KP, Kadam PH, Singh MK, Manik RS, Singla SK, Palta P, Chauhan MS. In vitro culture and morphological characterization of prepubertal buffalo (Bubalus bubalis) putative spermatogonial stem cell. J Assist Reprod Genet. 2012;29(12):1335-42. http://dx.doi.org/10.1007/s10815-012-9883-y. PMid:23151879.

Koga T, Koga T, Awai M, Tsutsui J, Yue BYJT, Tanihara H. Rho-associated protein kinase inhibitor, Y-27632, induces alterations in adhesion, contraction and motility in cultured human trabecular meshwork cells. Exp Eye Res. 2006;82(3):362-70. http://dx.doi.org/10.1016/j.exer.2005.07.006. PMid:16125171.

Kubota H, Avarbock MR, Brinster RL. Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells. Proc Natl Acad Sci USA. 2004;101(47):16489-94. http://dx.doi.org/10.1073/pnas.0407063101. PMid:15520394.

Kurosawa H. Application of Rho-associated protein kinase (ROCK) inhibitor to human pluripotent stem cells. J Biosci Bioeng. 2012;114(6):577-81. http://dx.doi.org/10.1016/j.jbiosc.2012.07.013. PMid:22898436.

Kushki D, Azarnia M, Gholami MR. Antioxidant effects of selenium on seminiferous tubules of immature mice testis. Zahedan J Res Med Sci. 2015;17(12):29-33. http://dx.doi.org/10.17795/zjrms-5188.

Lee J, Park S, Roh S. Y-27632, A ROCK inhibitor, delays senescence of putative murine salivary gland stem cells in culture. Arch Oral Biol. 2015;60(6):875-82. http://dx.doi.org/10.1016/j.archoralbio.2015.03.003. PMid:25804560.

Li X, Krawetz R, Liu S, Meng G, Rancourt DE. ROCK inhibitor improves survival of cryopreserved serum/feeder-free single human embryonic stem cells. Hum Reprod. 2009;24(3):580-9. http://dx.doi.org/10.1093/humrep/den404. PMid:19056770.

Liao JK, Seto M, Noma K. Rho kinase (ROCK) inhibitors. J Cardiovasc Pharmacol. 2007;50(1):17-24. http://dx.doi.org/10.1097/FJC.0b013e318070d1bd. PMid:17666911.

Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real time quantitative PCR and the 2(-Delta Delta C(T)). Method. Methods. 2001;25(4):402-8. http://dx.doi.org/10.1006/meth.2001.1262. PMid:11846609.

Mohapatra C, Barman HK, Panda RP, Kumar S, Das V, Mohanta R, Mohapatra SD, Jayasankar P. Cloning of cDNA and prediction of peptide structure of Plzf expressed in the spermatogonial cells of Labeo rohita. Mar Genomics. 2010;3(3-4):157-63. http://dx.doi.org/10.1016/j.margen.2010.09.002. PMid:21798210.

Nam J, Johnson J, Lannutti JJ, Agarwal S. Modulation of embryonic mesenchymal progenitor cell differentiation via control over pure mechanical modulus in electrospun nanofibers. Acta Biomater. 2011;7(4):1516-24. http://dx.doi.org/10.1016/j.actbio.2010.11.022. PMid:21109030.

Oatley JM, Avarbock MR, Telaranta AI, Fearon DT, Brinster RL. Identifying genes important for spermatogonial stem cell self-renewal and survival. Proc Natl Acad Sci USA. 2006;103(25):9524-9. http://dx.doi.org/10.1073/pnas.0603332103. PMid:16740658.

Panda RP, Barman HK, Mohapatra C. Isolation of enriched carp spermatogonial stem cells from Labeo rohita testis for in vitro propagation. Theriogenology. 2011;76(2):241-51. http://dx.doi.org/10.1016/j.theriogenology.2011.01.031. PMid:21496900.

Pertz O. Spatio-temporal Rho GTPase signaling - where are we now? J Cell Sci. 2010;123(Pt 11):1841-50. http://dx.doi.org/10.1242/jcs.064345. PMid:20484664.

Pirone DM, Liu WF, Ruiz SA, Gao L, Raghavan S, Lemmon CA, Romer LH, Chen CS. An inhibitory role for FAK in regulating proliferation: a link between limited adhesion and RhoA-ROCK signaling. J Cell Biol. 2006;174(2):277-88. http://dx.doi.org/10.1083/jcb.200510062. PMid:16847103.

Pramod RK, Mitra A. In vitro culture and characterization of spermatogonial stem cells on sertoli cell feeder layer in goat (Capra hircus). J Assist Reprod Genet. 2014;31(8):993-1001. http://dx.doi.org/10.1007/s10815-014-0277-1. PMid:24958548.

Qasemi-Panahi B, Movahedin M, Moghaddam G, Tajik P, Koruji M, Ashrafi-Helan J, Rafat SA. Isolation and proliferation of spermatogonial cells from ghezel sheep. Avicenna J Med Biotechnol. 2018;10(2):93-7. PMid:29849985.

Reid A, Gould A, Brand N, Cook M, Strutt P, Li J, Licht J, Waxman S, Krumlauf R, Zelent A. Leukemia translocation gene, PLZF, is expressed with a speckled nuclear pattern in early hematopoietic progenitors. Blood. 1995;86(12):4544-52. http://dx.doi.org/10.1182/blood.V86.12.4544.bloodjournal86124544. PMid:8541544.

Riento K, Ridley AJ. Rocks: multifunctional kinases in cell behavior. Nat Rev Mol Cell Biol. 2003;4(6):446-56. http://dx.doi.org/10.1038/nrm1128. PMid:12778124.

Rodriguez-Sosa JR, Dobson H, Hahnel A. Isolation and transplantation of spermatogonia in sheep. Theriogenology. 2006;66(9):2091-103. http://dx.doi.org/10.1016/j.theriogenology.2006.03.039. PMid:16870245.

Sahare MG, Suyatno S, Imai H. Recent advances of in vitro culture systems for spermatogonial stem cells in mammals. Reprod Med Biol. 2018;17(2):134-42. http://dx.doi.org/10.1002/rmb2.12087. PMid:29692670.

Shams A, Eslahi N, Movahedin M, Izadyar F, Asgari H, Koruji M. Future of spermatogonial stem cell culture: application of nanofiber scaffolds. Curr Stem Cell Res Ther. 2017;12(7):544-53. http://dx.doi.org/10.2174/1574888X12666170623095457. PMid:28641554.

Silva FM, Marques A, Chaveiro A. Reactive oxygen species: a double-edged sword in reproduction. Open Vet Sci J. 2010;4(1):127-33. http://dx.doi.org/10.2174/1874318801004010127.

Tsujimoto Y, Cossman J, Jaffe E, Croce CM. Involvement of the bcl-2 gene in human follicular lymphoma. Science. 1985;228(4706):1440-3. http://dx.doi.org/10.1126/science.3874430. PMid:3874430.

Wang J, Cao H, Xue X, Fan C, Fang F, Zhou J, Zhang Y, Zhang X. Effect of vitamin C on growth of caprine spermatogonial stem cells in vitro. Theriogenology. 2014;81(4):545-55. http://dx.doi.org/10.1016/j.theriogenology.2013.11.007. PMid:24368149.

Watanabe K, Ueno M, Kamiya D, Nishiyama A, Matsumura M, Wataya T, Takahashi JB, Nishikawa S, Nishikawa SH, Muguruma K, Sasai Y. A ROCK inhibitor permits survival of dissociated human Embryonic stem cells. Nat Biotechnol. 2007;25(6):681-6. http://dx.doi.org/10.1038/nbt1310. PMid:17529971.

Zheng Y, Zhang Y, Qu R, He Y, Tian X, Zeng W. Spermatogonial stem cells from domestic animals: progress and prospects. Reproduction. 2014;147(3):R65-74. http://dx.doi.org/10.1530/REP-13-0466. PMid:24357661.
 


Submitted date:
01/07/2021

Accepted date:
11/19/2021

61d592faa95395060163e984 animreprod Articles
Links & Downloads

Anim Reprod

Share this page
Page Sections