Animal Reproduction (AR)
https://animal-reproduction.org/article/doi/10.1590/1984-3143-AR2025-0162
Animal Reproduction (AR)
ORIGINAL ARTICLE

Spermatogonial stem cell markers and distribution in different regions of the testis of adult Nile tilapia (Oreochromis niloticus)

Sunny Okechukwu Abarikwu; Samyra Maria dos Santos Nassif Lacerda; Guilherme Mattos Jardim Costa; Luiz Renato de França

Downloads: 0
Views: 27

Abstract

Highly conserved vertebrate molecular markers, such as Gfra1, Pou5f3, Notch1, Plzf, and Nanos2, represent important candidates for evaluating spermatogonial stem cells (SSCs) physiology and preferential location in the fish testis. In tilapia, germ cell cysts are distributed within seminiferous tubules oriented along the dorso–ventral axis, while the adult testis continues to grow along both the longitudinal (cranio–caudal) and dorso–ventral axes. Herein we investigated the spatial distribution of undifferentiated spermatogonia (Aund) expressing established SSC-associated markers in the testes of sexually mature Nile tilapia. Topographical analysis along the longitudinal axis showed that Aund were predominantly located in regions of the seminiferous tubules adjacent to the interstitial compartment, with approximately 53% of Aund in the caudal region and 18% in the cranial region of the testis. Accordingly, most Aund positive for Gfra1, Plzf, and Nanos2 (>70%; p<0.05) were preferentially detected in the caudal region. Analysis along the dorso–ventral axis showed that Aund positive for Plzf, Gfrα1, Nanos2, Nanos1, Notch1, and Notch3 were more frequently observed in areas closer to the tunica albuginea compared with regions adjacent to the efferent ducts. Together, these findings demonstrate pronounced regional differences in the distribution of Aund along both the cranio–caudal and dorso–ventral axes of the Nile tilapia testis, highlighting a spatially organized germinal architecture that may be relevant for the maintenance of the germinal epithelium and post-pubertal testicular growth. To our knowledge, this is the first study to systematically describe the spatial distribution of Aund-associated markers in a teleost species.

Keywords

Tilapia (O. niloticus), spermatogonial stem cells (SSCs), SSCs niche, SSCs markers, testicular development

References

Ahmed SM, Hordofa B, Meressa BH, Tamiru M. Population structure and genetic diversity of Nile tilapia (Oreochromis niloticus) using microsatellite markers from selected water bodies in southwest Ethiopia. Vet Med Sci. 2023;9(5):2095-106. https://doi.org/10.1002/vms3.1212. PMid:37483169.

Alvarenga ER, França LR. Effects of different temperatures on testis structure and function with emphasis on somatic cells in sexually mature Nile tilapia (Oreochromis niloticus). Biol Reprod. 2009;80(3):537-44. https://doi.org/10.1095/biolreprod.108.072827. PMid:19020298.

Aoki Y, Nakamura S, Ishikawa Y, Tanaka M. Expression and syntenic analyses of four nanos genes in medaka. Zool Sci. 2009;26(2):112-8. https://doi.org/10.2108/zsj.26.112. PMid:19341327.

Batlouni SR, Nóbrega RH, França LR. Cell junctions in fish seminiferous epithelium. Fish Physiol Biochem. 2009;35(1):207-17. https://doi.org/10.1007/s10695-008-9253-y. PMid:18803027.

Bellaiche J, Lareyre JJ, Cauty C, Yano A, Allemand I, Le Gac F. Spermatogonial stem cell quest: nanos2, marker of a subpopulation of undifferentiated A spermatogonia in trout testis. Biol Reprod. 2014;90(4):79. https://doi.org/10.1095/biolreprod.113.116392. PMid:24554733.

Blanes-García M, Marinović Z, Herranz-Jusdado JG, Xie X, Ferrão L, Gallego V, Pérez L, Baloch AR, Horváth Á, Pšenička M, Asturiano JF, Morini M. Characterization of potential spermatogonia biomarker genes in the European eel (Anguilla anguilla). Fish Physiol Biochem. 2024;50(5):2099-115. https://doi.org/10.1007/s10695-024-01338-1. PMid:38639895.

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. https://doi.org/10.1530/REP-13-0316. PMid:24123129.

Cai Y, Wen J, Liu H, Wei R, Li X, Dong Y, Cheng K, Zou K. SMAD3 and HIF-1α orchestrate metabolic transition to aerobic glycolysis as a critical prerequisite for spontaneous reprogramming of spermatogonial stem cells. Stem Cell Res Ther. 2025;16(1):411. https://doi.org/10.1186/s13287-025-04541-w. PMid:40721814.

Chen L, Tang L, Lin Q, Zhang Y. Seahorse nanos3 plays essential roles in germ cell development in the absence of nanos. Water Biol Secur. 2025;4(1):100312. https://doi.org/10.1016/j.watbs.2024.100312.

de Rooij DG, Russell LD. All you wanted to know about spermatogonia but were afraid to ask. J Androl. 2000;21(6):776-98. https://doi.org/10.1002/j.1939-4640.2000.tb03408.x. PMid:11105904.

DeFalco T, Potter SJ, Williams AV, Waller B, Kan MJ, Capel B. Macrophages contribute to the spermatogonial niche in the adult testis. Cell Rep. 2015;12(7):1107-19. https://doi.org/10.1016/j.celrep.2015.07.015. PMid:26257171.

Dequéant ML, Pourquié O. Segmental patterning of the vertebrate embryonic axis. Nat Rev Genet. 2008;9(5):370-82. https://doi.org/10.1038/nrg2320. PMid:18414404.

Diao L, Turek PJ, John CM, Fang F, Reijo Pera RA. Roles of spermatogonial stem cells in spermatogenesis and fertility restoration. Front Endocrinol. 2022;13:895528. https://doi.org/10.3389/fendo.2022.895528. PMid:35634498.

Doretto LB, Butzge AJ, Nakajima RT, Martinez ERM, Souza BM, Rodrigues MDS, Rosa IF, Ricci JMB, Tovo-Neto A, Costa DF, Malafaia G, Shao C, Nóbrega RH. GDNF acts as a germ cell-derived growth factor and regulates the zebrafish germ stem cell niche in autocrine- and paracrine-dependent manners. Cells. 2022;11(8):1295. https://doi.org/10.3390/cells11081295. PMid:35455974.

Draper BW, McCallum CM, Moens CB. Nanos1 is required to maintain oocyte production in adult zebrafish. Dev Biol. 2007;305(2):589-98. https://doi.org/10.1016/j.ydbio.2007.03.007. PMid:17418113.

Duan L, Du S, Wang X, Zhou L, Liu Q, Li J. Glial cell line-derived neurotrophic factor (GDNF) is essential for colonization and expansion of turbot (Scophthalmus maximus) germ cells in recipients and in vitro culture. Theriogenology. 2024;214:1-9. https://doi.org/10.1016/j.theriogenology.2023.09.013. PMid:37837722.

Encinas G, Zogbi C, Stumpp T. Detection of four germ cell markers in rats during testis morphogenesis: differences and similarities with mice. Cells Tissues Organs. 2012;195(5):443-55. https://doi.org/10.1159/000329245. PMid:21893932.

França LR, Nobrega RH, Morais RDVS, De Castro Assis LH, Schulz RW. Sertoli cell structure and function in anamniote vertebrates. In: Griswold MD, editor. Sertoli cell biology. 2nd ed. London: Academic Press; 2015. p. 385-407. https://doi.org/10.1016/B978-0-12-417047-6.00013-2.

Froschauer A, Khatun MM, Sprott D, Franz A, Rieger C, Pfennig F, Gutzeit HO. Oct4-EGFP reporter gene expression marks the stem cells in embryonic development and in adult gonads of transgenic medaka. Mol Reprod Dev. 2013;80(1):48-58. https://doi.org/10.1002/mrd.22135. PMid:23139203.

Gao J, Wang J, Jiang J, Fan L, Wang W, Liu J, Zhang Q, Wang X. Identification and characterization of a nanog homolog in Japanese flounder (Paralichthys olivaceus). Gene. 2013;531(2):411-21. https://doi.org/10.1016/j.gene.2013.08.030. PMid:24013085.

Gao J, Wang X, Zhang Q. Evolutionary conservation of pou5f3 genomic organization and its dynamic distribution during embryogenesis and in adult gonads in Japanese flounder (Paralichthys olivaceus). Int J Mol Sci. 2017;18(1):231. https://doi.org/10.3390/ijms18010231. PMid:28124980.

Garcia TX, DeFalco T, Capel B, Hofmann MC. Constitutive activation of NOTCH1 signaling in Sertoli cells causes gonocyte exit from quiescence. Dev Biol. 2013;377(1):188-201. https://doi.org/10.1016/j.ydbio.2013.01.031. PMid:23391689.

Gautier A, Bosseboeuf A, Auvray P, Sourdaine P. Maintenance of potential spermatogonial stem cells in vitro by GDNF treatment in a chondrichthyan model (Scyliorhinus canicula L.). Biol Reprod. 2014;91(4):91-105. https://doi.org/10.1095/biolreprod.113.116020. PMid:25143357.

Glauche I, Moore K, Thielecke L, Horn K, Loeffler M, Roeder I. Stem cell proliferation and quiescence: two sides of the same coin. PLOS Comput Biol. 2009;5(7):e1000447. https://doi.org/10.1371/journal.pcbi.1000447. PMid:19629161.

Grisanti L, Falciatori I, Grasso M, Dovere L, Fera S, Muciaccia B, Fuso A, Berno V, Boitani C, Stefanini M, Vicini E. Identification of spermatogonial stem cell subsets by morphological analysis and prospective isolation. Stem Cells. 2009;27(12):3043-52. https://doi.org/10.1002/stem.206. PMid:19711452.

Hayashi T, Yamada T, Kageyama Y, Kihara K. Expression failure of the Notch signaling system is associated with the pathogenesis of testicular germ cell tumor. Tumour Biol. 2004;25(3):99-105. https://doi.org/10.1159/000079140. PMid:15361705.

Hayman ES, Fairgrieve WT, Luckenbach JA. Molecular and morphological sex differentiation in sablefish (Anoplopoma fimbria), a marine teleost with XX/XY sex determination. Gene. 2021;764:145093. https://doi.org/10.1016/j.gene.2020.145093. PMid:32866588.

Ikeda M, Kobayashi K, Nakayama-Sadakiyo Y, Sato Y, Tobita A, Saito M, Yamasu K. Transcriptome analysis suggested striking transition around the end of epiboly in the gene regulatory network downstream of the Oct4-type POU gene in zebrafish embryos. Dev Growth Differ. 2025;67(5):245-69. https://doi.org/10.1111/dgd.70012. PMid:40490365.

Kefi AS, Kang’Ombe J, Kassam D, Katongo C. Growth, reproduction and sex ratios in Oreochromis andersonii (Castelnau, 1861) fed with varying levels of 17α-methyltestosterone. J Aquac Res Dev. 2012;3:8.

Khanehzad M, Abbaszadeh R, Holakuyee M, Modarressi MH, Nourashrafeddin SM. FSH regulates RA signaling to commit spermatogonia into differentiation pathway and meiosis. Reprod Biol Endocrinol. 2021;19(1):4. https://doi.org/10.1186/s12958-020-00686-w. PMid:33407539.

Kostereva N, Hofmann MC. Regulation of the spermatogonial stem cell niche. Reprod Domest Anim. 2008;43(Suppl 2):386-92. https://doi.org/10.1111/j.1439-0531.2008.01189.x. PMid:18638151.

Lacerda SM, Costa GM, França LR. Biology and identity of fish spermatogonial stem cell. Gen Comp Endocrinol. 2014;207:56-65. https://doi.org/10.1016/j.ygcen.2014.06.018. PMid:24967950.

Lacerda SM, Costa GM, Silva MA, Campos-Junior PH, Segatelli TM, Peixoto MT, Resende RR, de França LR. Phenotypic characterization and in vitro propagation and transplantation of the Nile tilapia (Oreochromis niloticus) spermatogonial stem cells. Gen Comp Endocrinol. 2013;192:95-106. https://doi.org/10.1016/j.ygcen.2013.06.013. PMid:23792279.

Lacerda SMSN, Martinez ERM, Mura ILDD, Doretto LB, Costa GMJ, Silva MA, Digmayer M, Nóbrega RH, França LR. Duration of spermatogenesis and identification of spermatogonial stem cell markers in a Neotropical catfish, Jundiá (Rhamdia quelen). Gen Comp Endocrinol. 2019;273:249-59. https://doi.org/10.1016/j.ygcen.2018.10.018. PMid:30391241.

Leal MC, Cardoso ER, Nóbrega RH, Batlouni SR, Bogerd J, França LR, Schulz RW. Histological and stereological evaluation of zebrafish (Danio rerio) spermatogenesis with an emphasis on spermatogonial generations. Biol Reprod. 2009;81(1):177-87. https://doi.org/10.1095/biolreprod.109.076299. PMid:19339708.

Liu G, Luo F, Song Q, Wu L, Qiu Y, Shi H, Wang D, Zhou L. Blocking of progestin action disrupts spermatogenesis in Nile tilapia (Oreochromis niloticus). J Mol Endocrinol. 2014;53(1):57-70. https://doi.org/10.1530/JME-13-0300. PMid:24827000.

Liu L, Li J, Zhao C, Qu X, Liu X, Wang D, Wei J. The cellular expression patterns of gdnfa and gdnfb in the gonads of Nile tilapia and their differential response to retinoic acid. Theriogenology. 2024;224:1-8. https://doi.org/10.1016/j.theriogenology.2024.05.001. PMid:38714023.

Maekawa M, Saito S, Isobe D, Takemoto K, Miura Y, Dobashi Y, Yamasu K. The Oct4-related PouV gene, pou5f3, mediates isthmus development in zebrafish by directly and dynamically regulating pax2a. Cells Dev. 2024;179:203933. https://doi.org/10.1016/j.cdev.2024.203933. PMid:38908828.

Manosroi J, Petchjul K, Manosroi A. Effect of fluoxymesterone fish feed granule on sex reversal of the hybrid, Thai red tilapia (Oreochromis mossambicus Linn). Asian Fish Sci. 2004;17(4):323-31. https://doi.org/10.33997/j.afs.2004.17.4.005.

Mohapatra C, Barman HK. Identification of promoter within the first intron of Plzf gene expressed in carp spermatogonial stem cells. Mol Biol Rep. 2014;41(10):6433-40. https://doi.org/10.1007/s11033-014-3525-7. PMid:24990695.

Nakajima S, Hayashi M, Kouguchi T, Yamaguchi K, Miwa M, Yoshizaki G. Expression patterns of gdnf and gfrα1 in rainbow trout testis. Gene Expr Patterns. 2014;14(2):111-20. https://doi.org/10.1016/j.gep.2014.01.006. PMid:24518650.

Nakamura S, Kobayashi K, Nishimura T, Higashijima S, Tanaka M. Identification of germline stem cells in the ovary of the teleost medaka. Science. 2010;328(5985):1561-3. https://doi.org/10.1126/science.1185473. PMid:20488987.

National Institutes of Health – NIH [homepage on the Internet]. Former Home of ImageJ. 2025 [cited 2025 Oct 28]. Available from: http://rsbweb.nih.gov/ij

Naughton CK, Jain S, Strickland AM, Gupta A, Milbrandt J. Glial cell line-derived neurotrophic factor-mediated RET signaling regulates spermatogonial stem cell fate. Biol Reprod. 2006;74(2):314-21. https://doi.org/10.1095/biolreprod.105.047365. PMid:16237148.

Nóbrega RH, Greebe CD, van de Kant H, Bogerd J, França LR, Schulz RW. Spermatogonial stem cell niche and spermatogonial stem cell transplantation in zebrafish. PLoS One. 2010;5(9):e12808. https://doi.org/10.1371/journal.pone.0012808. PMid:20862221.

Ozaki Y, Saito K, Shinya M, Kawasaki T, Sakai N. Evaluation of Sycp3, Plzf and Cyclin B3 expression and suitability as spermatogonia and spermatocyte markers in zebrafish. Gene Expr Patterns. 2011;11(5-6):309-15. https://doi.org/10.1016/j.gep.2011.03.002. PMid:21402175.

Palladino A, De Felice E, Attanasio C, Barone CMA, Crasto A, D’Angelo L, Giaquinto D, Lambiase C, Scocco P, Serrapica F, Maruccio L. A morphological and ultrastructural study of the anterior digestive tract of adult Nile tilapia Oreochromis niloticus. Animals. 2023;13(3):420. https://doi.org/10.3390/ani13030420. PMid:36766309.

Pfennig F, Kurth T, Meißner S, Standke A, Hoppe M, Zieschang F, Reitmayer C, Gobel A, Kretzschmar G, Gutzeit HO. The social status of the male Nile tilapia (Oreochromis niloticus) influences testis structure and gene expression. Reproduction. 2012;143(1):71-84. https://doi.org/10.1530/REP-11-0292. PMid:22031714.

Qian P, Kang J, Liu D, Xie G. Single-cell transcriptome sequencing of zebrafish testis revealed novel spermatogenesis marker genes and stronger Leydig–germ cell paracrine interactions. Front Genet. 2022;13:851719. https://doi.org/10.3389/fgene.2022.851719. PMid:35360857.

Rajachandran S, Zhang X, Cao Q, Caldeira-Brant AL, Zhang X, Song Y, Evans M, Bukulmez O, Grow EJ, Nagano M, Orwig KE, Chen H. Dissecting the spermatogonial stem cell niche using spatial transcriptomics. Cell Rep. 2023;42(7):112737. https://doi.org/10.1016/j.celrep.2023.112737. PMid:37393620.

Sada A, Hasegawa K, Pin PH, Saga Y. NANOS2 acts downstream of glial cell line-derived neurotrophic factor signaling to suppress differentiation of spermatogonial stem cells. Stem Cells. 2012;30(2):280-91. https://doi.org/10.1002/stem.790. PMid:22102605.

Sambe N, Yoshihara M, Nishino T, Sugiura R, Nakayama T, Louis C, Takahashi S. Analysis of Notch1 signaling in mammalian sperm development. BMC Res Notes. 2023;16(1):108. https://doi.org/10.1186/s13104-023-06378-z. PMid:37337280.

Sánchez-Sánchez AV, Camp E, García-España A, Leal-Tassias A, Mullor JL. Medaka Oct4 is expressed during early embryo development, and in primordial germ cells and adult gonads. Dev Dyn. 2010;239(2):672-9. https://doi.org/10.1002/dvdy.22198. PMid:20034054.

Schulz RW, França LR, Lareyre JJ, Legac F, Chiarini-Garcia H, Nóbrega RH, Miura T. Spermatogenesis in fish. Gen Comp Endocrinol. 2010;165(3):390-411. https://doi.org/10.1016/j.ygcen.2009.02.013. PMid:19348807.

Schulz RW, Menting S, Bogerd J, França LR, Vilela DAR, Godinho HP. Sertoli cell proliferation in the adult testis: evidence from two fish species belonging to different orders. Biol Reprod. 2005;73(5):891-8. https://doi.org/10.1095/biolreprod.105.039891. PMid:16000552.

Setthawong P, Khemthong M, Lertwanakarn T, Surachetpong W. Ultrasonography for non-invasive sex identification and reproductive assessment in Nile tilapia (Oreochromis niloticus). Front Vet Sci. 2024;11:1467158. https://doi.org/10.3389/fvets.2024.1467158. PMid:39444737.

Shang M, Su B, Lipke EA, Perera DA, Li C, Qin Z, Li Y, Dunn DA, Cek S, Peatman E, Dunham RA. Spermatogonial stem cells specific marker identification in channel catfish, Ictalurus punctatus, and blue catfish, I. furcatus. Fish Physiol Biochem. 2015;41(6):1545-56. https://doi.org/10.1007/s10695-015-0106-1. PMid:26251285.

Siqueira-Silva DH, Santos Silva AP, Silva Costa R, Senhorini JA, Ninhaus-Silveira A, Verissimo-Silveira R. Preliminary study on testicular germ cell isolation and transplantation in an endangered endemic species Brycon orbignyanus (Characiformes: characidae). Fish Physiol Biochem. 2021;47(3):767-76. https://doi.org/10.1007/s10695-019-00631-8. PMid:30937624.

Takehashi M, Tada M, Kanatsu-Shinohara M, Morimoto H, Kazuki Y, Oshimura M, Tada T, Shinohara T. Hybridization of testis-derived stem cells with somatic cells and embryonic stem cells in mice. Biol Reprod. 2012;86(6):178. https://doi.org/10.1095/biolreprod.112.098988. PMid:22441799.

Thönnes M, Prause R, Levavi-Sivan B, Pfennig F. Transcriptomes of testis and pituitary from male Nile tilapia (O. niloticus L.) in the context of social status. PLoS One. 2022;17(5):e0268140. https://doi.org/10.1371/journal.pone.0268140. PMid:35544481.

Voigt AL, Lima EMN, Lara N, Dobrinski I. Comparing the adult and pre-pubertal testis: metabolic transitions and the change in the spermatogonial stem cell metabolic microenvironment. Andrology. 2023;11(6):1132-46. https://doi.org/10.1111/andr.13397. PMid:36690000.

Voronina A, Pshennikova E. The Vox mRNA and protein expression in zebrafish Pou5f3 MZspg mutant embryos. Stem Cell Investig. 2016;3:79. https://doi.org/10.21037/sci.2016.11.01. PMid:28066781.

Wang D, Manali D, Wang T, Bhat N, Hong N, Li Z, Wang L, Yan Y, Liu R, Hong Y. Identification of pluripotency genes in the fish medaka. Int J Biol Sci. 2011;7(4):440-51. https://doi.org/10.7150/ijbs.7.440. PMid:21547061.

Xie X, Nóbrega R, Pšenička M. Spermatogonial stem cells in fish: characterization, isolation, enrichment, and recent advances of in vitro culture systems. Biomolecules. 2020;10(4):644. https://doi.org/10.3390/biom10040644. PMid:32331205.

Yoshida S. Elucidating the identity and behavior of spermatogonial stem cells in the mouse testis. Reproduction. 2012;144(3):293-302. https://doi.org/10.1530/REP-11-0320. PMid:22733803.

Zhong C, Liu M, Tao Y, Wu X, Yang Y, Wang T, Meng Z, Xu H, Liu X. Pou5f1 and Nanog are reliable germ cell-specific genes in gonad of a protogynous hermaphroditic fish, orange-spotted grouper (Epinephelus coioides). Genes. 2021;13(1):79. https://doi.org/10.3390/genes13010079. PMid:35052423.
 


Submitted date:
10/28/2025

Accepted date:
05/05/2026

6a636d46a953956f5278c524 animreprod Articles
Links & Downloads

Anim Reprod

Share this page
Page Sections