Nejvíce citovaný článek - PubMed ID 22219211
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 plzf, dnd, boule, and kitr were significantly higher in type A spermatogonia than in later developed germ cells. In addition, plzf 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 plzf 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.
- Klíčová slova
- PLZF, fluorescence-activated cell sorting, germ stem cell, gonad, spermatogonia, sturgeon,
- Publikační typ
- časopisecké články MeSH
Spermatogenesis is a continuous and dynamic developmental process, in which a single diploid spermatogonial stem cell (SSC) proliferates and differentiates to form a mature spermatozoon. Herein, we summarize the accumulated knowledge of SSCs and their distribution in the testes of teleosts. We also reviewed the primary endocrine and paracrine influence on spermatogonium self-renewal vs. differentiation in fish. To provide insight into techniques and research related to SSCs, we review available protocols and advances in enriching undifferentiated spermatogonia based on their unique physiochemical and biochemical properties, such as size, density, and differential expression of specific surface markers. We summarize in vitro germ cell culture conditions developed to maintain proliferation and survival of spermatogonia in selected fish species. In traditional culture systems, sera and feeder cells were considered to be essential for SSC self-renewal, in contrast to recently developed systems with well-defined media and growth factors to induce either SSC self-renewal or differentiation in long-term cultures. The establishment of a germ cell culture contributes to efficient SSC propagation in rare, endangered, or commercially cultured fish species for use in biotechnological manipulation, such as cryopreservation and transplantation. Finally, we discuss organ culture and three-dimensional models for in vitro investigation of fish spermatogenesis.
- Klíčová slova
- fish, florescence-activated cell sorting (FACS), germ cell culture, magnetic-activated cell sorting (MACS), spermatogenesis, spermatogonial stem cell (SSC),
- MeSH
- buněčné kultury * MeSH
- kmenové buňky cytologie ultrastruktura MeSH
- ryby metabolismus MeSH
- separace buněk * MeSH
- spermatogeneze MeSH
- spermatogonie cytologie ultrastruktura MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
We are entering an exciting epoch in livestock biotechnology during which the fundamental approaches (such as transgenesis, spermatozoa cryopreservation and artificial insemination) will be enhanced based on the modern understanding of the biology of spermatogonial stem cells (SSCs) combined with the outstanding recent advances in genomic editing technologies and in vitro cell culture systems. The general aim of this review is to outline comprehensively the promising applications of SSC manipulation that could in the nearest future find practical application in livestock breeding. Here, we will focus on 1) the basics of mammalian SSC biology; 2) the approaches for SSC isolation and purification; 3) the available in vitro systems for the stable expansion of isolated SSCs; 4) a discussion of how the manipulation of SSCs can accelerate livestock transgenesis; 5) a thorough overview of the techniques of SSC transplantation in livestock species (including the preparation of recipients for SSC transplantation, the ultrasonographic-guided SSC transplantation technique in large farm animals, and the perspectives to improve further the SSC transplantation efficiency), and finally, 6) why SSC transplantation is valuable to extend the techniques of spermatozoa cryopreservation and/or artificial insemination. For situations where no reliable data have yet been obtained for a particular livestock species, we will rely on the data obtained from studies conducted in rodents because the knowledge gained from rodent research is translatable to livestock species to a great extent. On the other hand, we will draw special attention to situations where such translation is not possible.
