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

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 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.

Keywords: PLZF; fluorescence-activated cell sorting; germ stem cell; gonad; spermatogonia; sturgeon.

FIGURE 1

Histological observation and light scattering property-dependent fractionation of 6 month-old sterlet testes. (A) Testes section stained with hematoxylin and eosin (HE). (B) Testes section stained with anti-vasa antibody. (C) The proportion of germ cells and somatic cells in testes. ASG, type A spermatogonia. (D, E) Bivariate histograms of flow cytometry data from dissociated testicular cells prepared from 6 month-old sterlets. Gates P1-P3 were set on the histogram and used for cell sorting. (F) Vasa-positive rates of sorted cells in gates P1-P3 and unsorted live cells [mean ± standard deviation of mean (SD)]. Means with an asterisk indicate a significant difference (n = 6, p < 0.05). (G) Microscopic observation of sorted cells using gates P1-P3 and HE stained cells from gate P1 (small rectangle). Immunofluorescent staining for detection/calculation of vasa positive cells in different gates. Blue signal, DAPI staining, Green signal, vasa antibody staining.

FIGURE 2

Histological observation and light scattering property-dependent fractionation of 18 month-old sterlet testes. (A) Testes sections stained with hematoxylin and eosin (HE). (B) Testes sections stained with anti-vasa antibody. (C) The proportion of various germ cell populations and somatic cells in testes. ASG, type A spermatogonia; BSG, type B spermatogonia. (D, E) Bivariate histograms of flow cytometry data from dissociated testicular cells prepared from 18 month-old sterlets. Gates P1-P3 were set on the histogram and used for cell sorting. (F) Vasa-positive rates of sorted cells in gates P1-P3 and unsorted live cells [mean ± standard deviation of mean (SD)]. Means with an asterisk indicate a significant difference (n = 6, p < 0.05). (G) Microscopic observation of sorted cells using gates P1-P3. (H) Cells from gates P1-P3 stained with anti-vasa antibody and DAPI.

FIGURE 3

Histological observation and light scattering property-dependent fractionation of 2 year-old sterlet testes. (A) Testes sections stained with hematoxylin and eosin (HE). (B) The proportion of various germ cell populations and somatic cells in testes. ASG, type A spermatogonia; BSG, type B spermatogonia; SPC, primary spermatocyte; SSC, secondary spermatocyte. (C, D) Bivariate histograms of flow cytometry data from dissociated testicular cells prepared from 2 year-old sterlets. Gates P1–P3 were set on the histogram and used for cell sorting. (E) Vasa-positive rates of sorted cells in gates P1–P3 and unsorted live cells [mean ± standard error of mean (SEM)]. Means with an asterisk indicate a significant difference (n = 5, p < 0.05). (F) Microscopic observation of sorted cells using gates P1–P3. (G) Cells from gates P1-P3 stained with anti-vasa antibody and DAPI.

FIGURE 4

Expression of candidate markers for sorted cell populations. (A) Comparision of germ cell related genes expression profile of P1 cells between stage I and II testes by q-PCR. Gene expressions in stage I were defined as the relative value 1.0. (B) Comparision of germ cell related gene expression profiles of P1 and P2 cells at stage II testes by q-PCR. Gene expressions in P1 were defined as the relative value 1.0. Cq values were normalized per gene for relative quantification using the arithmetic mean of differentiated group values (A) and P1 values (B). $Rq =2^{-(Cq- x^3)}$ . Data are presented as geometric mean value ± geometric standard deviation (GSD). Means with an asterisk indicate a significant difference (n = 3, p < 0.05). (C) Histological observation of early stage testes used for in situ hybridization (ISH). (D) The cellular distributions of plzf by ISH. (E) Merged photograph with DAPI. (F) Histological observation of testes at a later maturing stage used for ISH. (G) The cellular distributions of plzf by ISH. (H) Merged photograph with DAPI.