Isolation and Characterization of Highly Pure Type A Spermatogonia From Sterlet () Using Flow-Cytometric Cell Sorting

Overview

Journal Front Cell Dev Biol

Specialty Cell Biology

Date 2021 Dec 27

PMID 34957105

Citations 2

Authors

Xuan Xie

Tomas Tichopad

Galina Kislik

Lucie Langerova

Pavel Abaffy

Radek Sindelka

Roman Franek

Michaela Fucikova

Christoph Steinbach

Mujahid Ali Shah

Ivo Sauman

Fan Chen

Martin Psenicka

Affiliations

Soon will be listed here.

Abstract

Sturgeons are among the most ancient linages of actinopterygians. At present, many sturgeon species are critically endangered. Surrogate production could be used as an affordable and a time-efficient method for endangered sturgeons. Our study established a method for identifying and isolating type A spermatogonia from different developmental stages of testes using flow cytometric cell sorting (FCM). Flow cytometric analysis of a whole testicular cell suspension showed several well-distinguished cell populations formed according to different values of light scatter parameters. FCM of these different cell populations was performed directly on glass slides for further immunocytochemistry to identify germ cells. Results showed that the cell population in gate P1 on a flow cytometry plot (with high forward scatter and high side scatter parameter values) contains the highest amount of type A spermatogonia. The sorted cell populations were characterized by expression profiles of 10 germ cell specific genes. The result confirmed that setting up for the P1 gate could precisely sort type A spermatogonia in all tested testicular developmental stages. The P2 gate, which was with lower forward scatter and side scatter values mostly, contained type B spermatogonia at a later maturing stage. Moreover, expressions of , and were significantly higher in type A spermatogonia than in later developed germ cells. In addition, was firstly found as a reliable marker to identify type A spermatogonia, which filled the gap of identification of spermatogonial stem cells in sterlet. It is expected to increase the efficiency of germ stem cell culture and transplantation with identification. Our study thus first addressed a phenotypic characterization of a pure type A spermatogonia population in sterlet. FCM strategy can improve the production of sturgeons with surrogate broodstock and further the analysis of the cellular and molecular mechanisms of sturgeon germ cell development.

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References

Vizziano-Cantonnet D, Lasalle A, Di Landro S, Klopp C, Genthon C . De novo transcriptome analysis to search for sex-differentiation genes in the Siberian sturgeon. Gen Comp Endocrinol. 2018; 268:96-109. DOI: 10.1016/j.ygcen.2018.08.007. View

Berbejillo J, Martinez-Bengochea A, Bedo G, Brunet F, Volff J, Vizziano-Cantonnet D . Expression and phylogeny of candidate genes for sex differentiation in a primitive fish species, the Siberian sturgeon, Acipenser baerii. Mol Reprod Dev. 2012; 79(8):504-16. DOI: 10.1002/mrd.22053. View

Xie X, Li P, Psenicka M, Ye H, Steinbach C, Li C . Optimization of In Vitro Culture Conditions of Sturgeon Germ Cells for Purpose of Surrogate Production. Animals (Basel). 2019; 9(3). PMC: 6466142. DOI: 10.3390/ani9030106. View

Schulz R, de Franca L, Lareyre J, Le Gac F, LeGac F, Chiarini-Garcia H . Spermatogenesis in fish. Gen Comp Endocrinol. 2009; 165(3):390-411. DOI: 10.1016/j.ygcen.2009.02.013. View

Fajkowska M, Rzepkowska M, Adamek D, Ostaszewska T, Szczepkowski M . Expression of dmrt1 and vtg genes during gonad formation, differentiation and early maturation in cultured Russian sturgeon Acipenser gueldenstaedtii. J Fish Biol. 2016; 89(2):1441-9. DOI: 10.1111/jfb.12992. View

Youngren K, Coveney D, Peng X, Bhattacharya C, Schmidt L, Nickerson M . The Ter mutation in the dead end gene causes germ cell loss and testicular germ cell tumours. Nature. 2005; 435(7040):360-4. PMC: 1421521. DOI: 10.1038/nature03595. View

Ye H, Li C, Yue H, Yang X, Wei Q . Differential expression of fertility genes boule and dazl in Chinese sturgeon (Acipenser sinensis), a basal fish. Cell Tissue Res. 2015; 360(2):413-25. DOI: 10.1007/s00441-014-2095-2. View

Schulz R, Menting S, Bogerd J, Franca L, Vilela D, Godinho H . Sertoli cell proliferation in the adult testis--evidence from two fish species belonging to different orders. Biol Reprod. 2005; 73(5):891-8. DOI: 10.1095/biolreprod.105.039891. View

Shang M, Su B, Lipke E, Perera D, Li C, Qin Z . Spermatogonial stem cells specific marker identification in channel catfish, Ictalurus punctatus and blue catfish, I. furcatus. Fish Physiol Biochem. 2015; 41(6):1545-56. DOI: 10.1007/s10695-015-0106-1. View

10.

Runyan C, Schaible K, Molyneaux K, Wang Z, Levin L, Wylie C . Steel factor controls midline cell death of primordial germ cells and is essential for their normal proliferation and migration. Development. 2006; 133(24):4861-9. DOI: 10.1242/dev.02688. View

11.

Ye H, Li C, Yue H, Du H, Yang X, Yoshino T . Establishment of intraperitoneal germ cell transplantation for critically endangered Chinese sturgeon Acipenser sinensis. Theriogenology. 2017; 94:37-47. DOI: 10.1016/j.theriogenology.2017.02.009. View

12.

Takeuchi Y, Yoshizaki G, Takeuchi T . Biotechnology: surrogate broodstock produces salmonids. Nature. 2004; 430(7000):629-30. DOI: 10.1038/430629a. View

13.

Morita T, Morishima K, Miwa M, Kumakura N, Kudo S, Ichida K . Functional Sperm of the Yellowtail (Seriola quinqueradiata) Were Produced in the Small-Bodied Surrogate, Jack Mackerel (Trachurus japonicus). Mar Biotechnol (NY). 2015; 17(5):644-54. DOI: 10.1007/s10126-015-9657-5. View

14.

Nayak S, Ferosekhan S, Sahoo S, Sundaray J, Jayasankar P, Barman H . Production of fertile sperm from in vitro propagating enriched spermatogonial stem cells of farmed catfish, Clarias batrachus. Zygote. 2016; 24(6):814-824. DOI: 10.1017/S0967199416000149. View

15.

Iwasaki-Takahashi Y, Shikina S, Watanabe M, Banba A, Yagisawa M, Takahashi K . Production of functional eggs and sperm from in vitro-expanded type A spermatogonia in rainbow trout. Commun Biol. 2020; 3(1):308. PMC: 7296041. DOI: 10.1038/s42003-020-1025-y. View

16.

Rolland A, Lareyre J, Goupil A, Montfort J, Ricordel M, Esquerre D . Expression profiling of rainbow trout testis development identifies evolutionary conserved genes involved in spermatogenesis. BMC Genomics. 2009; 10:546. PMC: 2786911. DOI: 10.1186/1471-2164-10-546. View

17.

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

18.

Franek R, Marinovic Z, Lujic J, Urbanyi B, Fucikova M, Kaspar V . Cryopreservation and transplantation of common carp spermatogonia. PLoS One. 2019; 14(4):e0205481. PMC: 6472724. DOI: 10.1371/journal.pone.0205481. View

19.

Aramaki S, Sato F, Kato T, Soh T, Kato Y, Hattori M . Molecular cloning and expression of dead end homologue in chicken primordial germ cells. Cell Tissue Res. 2007; 330(1):45-52. DOI: 10.1007/s00441-007-0435-1. View

20.

Zhang F, Li X, He M, Ye D, Xiong F, Amin G . Efficient generation of zebrafish maternal-zygotic mutants through transplantation of ectopically induced and Cas9/gRNA targeted primordial germ cells. J Genet Genomics. 2020; 47(1):37-47. DOI: 10.1016/j.jgg.2019.12.004. View