Cells in population 3 were larger than those in the other two populations. Because of the presence of more cytoplasm, their nuclei did not contain such a prominent nucleolus as populations 1 or 2, and these cells were typically found in groups at the periphery of the cord (labeled 3 in Fig. 3). Population 3 was particularly noticeable in sections taken from testes at 18 and 19 wk of gestation.
Identification of Germ Cell Subpopulations by Immunohistochemical Profiling
OCT 4. OCT4 was immunolocalized to the nuclei of the majority of germ cells in the testes from the first trimester (Fig. 4, a and b). In testes from the second trimester (Fig. 4, c and d), OCT4 immunopositive staining was restricted to a subset of germ cell nuclei that, based on their morphological appearance, were identified as belonging to those of population 1.
OCT 4/C-KIT. Dual immunohistochemistry revealed that OCT4-immunopositive cells (Fig. 4, e and f, blue nuclei) expressed C-KIT on their surface. Germ cells that were oCT4neg/C-KITneg were identified in the same sections (Fig. 4f, arrows).
CHK2/C-KIT. Three staining patterns were observed: CHK2pos/C-KITpos (Fig. 5, a and b, blue nuclei, brown membrane), CHK2pos/C-KITneg (Fig. 5b, inset), and CHK2neg/C-KITneg (Fig. 5a, arrows).
MAGE-A4/C-KIT. Costaining with antibodies against MAGE-A4 and C-KIT resulted in identification of separate groups of germ cells. Coexpression of the two proteins was not observed in either the first or second trimester (Fig. 5, c and d).
MAGE-A4/CHK2. CHK2 was detected in most germ cells (Fig. 6a), but coexpression of MAGE-A4 was only seen in a subset of these CHK2pos/MAGEpos) (Fig. 6a, arrows).
OCT4/MAGE-A4. The OCT4- and MAGE-A4-immuno-positive germ cell populations were distinct, and the two proteins were never coexpressed (Fig. 6, b and c).
CHK2/C-KIT/MAGE-A4. Triple-staining resulted in identification of three different types of CHK2 immunopositive cells: CHK2pos/C-KITpos (Fig. 6, day, e, asterisks), CHK2pos/MAGE-A4pos (Fig. 6, day, e, arrows), and CHK2pos/C-KITneg/MAGE-A4neg (Fig. 6e, blue nuclei with no membrane staining).
FIG. 5. Identification of subpopulations of germ cells in second-trimester testes based on immunoexpression of C-KIT, Chk-2, and MAGE-A4. Two subpopulations of CHK2-positive cells (blue nuclei) were identified: CHK2pos/C-KITneg and CHK2pos/ C-KITpos (a, b, and inset in b). Some cells were CHK2neg/C-KITneg (arrows in a). When sections were costained with antibodies directed against C-KIT (brown) and MAGE-A4 (purple), no cells expressing both germ cell markers were identified (c and d). Gestational ages: 14 wk (a and c), 19 wk (b), and 18 wk (d). Magnification, X100.
TABLE 3. Summary of the phenotypes of the three subpopulations of human fetal germ cells identified in second trimester samples.
a Based on information in Figure 3.
b Based on immunolocalisation of PCNA and phosphorylated histone H3 (unpublished results).
FIG. 6. Immunofluorescent staining of germ cell markers in second-trimester human fetal testis. a) CHK2 (blue) and MAGE-A4 (green) at 16 wk were coexpressed in a subpopulation of germ cells (arrows), but the majority of CHK2pos cells were immunonegative for MAGE-A4. b and c) At 17 wk, double-staining for OCT4 (red) and MAGE-A4 (blue, arrows in c) revealed that no germ cells coexpressed both proteins. d and e) At 16 wk and 19 wk, triple-staining was performed for C-KIT (red), CHK2 (blue), and MAGE-A4 (green). None of the germ cells coexpressed C-KIT and MAGE-A4; however, CHK2p°s/C-KITp“ (asterisks) and CHK2pos/ MAGE-A4pos (arrows in d, e) populations existed within the same seminiferous cord. Magnification, X40 (a and b) and X100 (c-e).