In Vitro Ectopic Behavior of Porcine Spermatogonial Germ Cells and Testicular Somatic Cells

Abstract

Embryonic body-like colony formation is a unique pattern in male germ cell cultures, including spermatogonial stem cells. However, detailed information of the colony formation has not yet been sufficiently reported in male germ cell culture. To elucidate the formation of germ cell-derived colony (GDC), glial cell-derived neurotrophic factor receptor alpha-1 (GFRα-1)-positive pig germ cells were isolated using an immunomagnetic cell isolation method and labeled with red- or green-fluorescent dye. In GDC culture, red-fluorescent-labeled germ cells were evenly distributed in the wells from day 1 to 4, and they clustered together at the time of GDC formation on day 6. Interestingly, feeder cells migrated to the site of colony formation as spermatogonia carriers. Furthermore, when freshly prepared green-labeled GFRα-1-positive germ cells were added, mixed-fluorescent dye (red and green) colonies were observed. On bromodeoxyuridine (BrdU) treatment, 58% ± 3.13% of germ cells were positive to protein gene product 9.5 but negative to BrdU cells. Immunocytochemistry and reverse transcription-polymerase chain reaction results showed that cultured GDC cells were positive to stem cell- and pig germ cell-specific marker genes. In conclusion, in vitro formation of GDCs is mainly dependent on the aggregation of single germ cells as well as on the slow proliferation of germ cells.

Previously claimed male germline stem cells from porcine testis are actually progenitor Leydig cells

“…Previous investigations have shown that mGSCs can be successfully isolated from mice [ 26 ], rats [ 27 ], pigs [ 28 ], and humans [ 29 ]. Cultured mouse mGSCs, however, exhibit different characteristics to those of human [ 30 , 31 ] and domestic animals, including pig [ 11 , 32 , 33 ], goat [ 12 , 34 , 35 ], cattle [ 13 , 36 ], and chicken [ 37 ]. The reason why mGSCs displayed distinct morphological differences among species remains unclear.…”