No staining was observed when the primary antibodies were replaced by normal rabbit serum (Fig. 1i, inset).
Differential Expression of Proteins by Germ Cells During the First and Second Trimesters
In first-trimester testes, expression of PLAP was detected in most germ cells (Fig. 2, day), whereas immunolocali-zation of CHK2 was variable between samples, with some being immunonegative. In those testes that were immuno-positive for CHK2 (e.g., Fig. 2a), most of the germ cells had intense immunopositive nuclear staining. Cells that were immunopositive for C-KIT (Fig. 2g, arrow), MAGE-A4 (Fig. 2j), and PCTAIRE-1 (not shown) were rare, and no immunopositive cells were detected in some samples.
During the second trimester, the proportions of cells that were immunopositive for the same panel of antibodies was dependent on the gestational age of the fetus (Table 2). For example, at 14 wk of gestation (Fig. 2, b, e, h, and k), most germ cells were immunopositive for CHK2, PCTAIRE-1, or C-KIT, but few cells were immunopositive for PLAP or MAGE-A4. However, by 19 wk of gestation (Fig. 2, c, f, i, and l), although many CHK2-positive cells were still present, cells that were immunopositive for C-KIT or PCTAI-RE-1 appeared to be reduced compared to 14 wk, and PLAP-immunopositive cells were rare.
FIG. 2. Comparative staining of germ cell populations within fetal testes during first and second trimester. Gestational ages of 7-9 wk (a, d, g, and j), 14 wk (b, e, h, and k), and 19 wk (c, f, i, and l). a-c) CHK2. d-f) PLAP. g and h) C-KIT. j-l) MAGE-A4. Note that images at 14 and 19 wk were taken from the same two testes in all panels to allow direct comparison between the proportions of germ cells stained for each protein at each age. Arrows indicate membrane-specific staining for PLAP (f) and C-KIT (g and i). Inset in c shows a typical control in which primary antibody was replaced by normal mouse serum. Magnification, X40.
TABLE 2. Summary of the proportion of germ cells immunopositive for selected marker proteins in testes from first and second trimester.a
a Maximum score is + + + +, indicating that all germ cells are positive; this score does not reflect the intensity of the immunopositive reaction. A score of (+) indicates that immunopositive cells were rare and not detected in every sample examined.
b Some first-trimester samples were immunonegative for CHK2.