Proliferation of germ cells and somatic cells in first trimester human embryonic gonads as indicated by S and S+G2+M phase fractions

Abstract

Objectives: The number of germ cells and somatic cells in human embryonic and foetal gonads has previously been estimated by stereological methods, which are time- and labour-consuming with little information concerning cell proliferation. Here, we studied whether flow cytometry could be applied as an easier method, also enabling estimation of the fraction of cells in S or S+G(2)+M (SG(2) M) cell-cycle phases as indicators of cell proliferation.

Methods: Cell suspensions from 35 human embryonic gonads at days 37 to 68 post-conception (pc) were immunomagnetically sorted into C-KIT positive (germ) cells and negative (somatic) cells. They were stained for DNA content and analysed by flow cytometry. S and SG(2) M fractions could be measured for 13 of the female and 20 of the male gonads. The number of cells was estimated using fluorescent reference beads.

Results: During the period from 37 to 68 days pc, female germ and somatic cells had a stable S and SG(2) M fractions indicating steady growth of both subpopulations, whereas they decreased in both male germ and somatic cells. The number of germ and somatic cells estimated by flow cytometry was significantly lower than in stereological estimates, suggesting loss of cells during preparation.

Conclusions: Cell proliferation as indicated by S and SG(2) M fractions could be estimated specifically for primordial germ and somatic cells. Estimation of total number of germ and somatic cells was not feasible.

Figure 1

Flow cytometry evaluation of immunomagnetic sorting of germ cells versus somatic cells. Cell suspensions were prepared from a set of mouse embryonic gonads (at 12½ days pc) and immunomagnetically sorted according to CD117 expression (clone AC126; Miltenyi Biotech). Sorted suspensions (except controls) were stained with phycoerythrin‐conjugated anti‐CD117 antibody (CD117‐PE; clone A3C6E2, Miltenyi Biotech). Analysis of CD117‐PE expression (lower panel) was performed on a subpopulation defined by forward and side scatter (R₁, upper panel). Estimates of CD117‐PE positive fraction are indicated (R₅, lower panel).

Figure 2

Alkaline phosphatase (AP) expression of human embryonic gonad cells in the CD117‐positive fraction from immunomagnetic sorting. Insert shows an AP positive cell at higher magnification. Male gonad, 53 days pc, patient no. 66.

Figure 3

Flow cytometry analysis of cell cycle distribution in a human embryonic gonad at 42 days pc. (a) Germ cells. (b) Somatic cells. Gonad cell suspensions were immunomagnetically sorted into CD117 positive (germ) cells and negative (somatic) cells, stained with propidium iodide, and analysed by flow cytometry. DNA fluorescence histograms, gated in forward versus side scatter (R₁) and fluorescence pulse area versus height (R₂), were deconvoluted into cell cycle fractions (G₁, S, G₂ + M). Nile red beads were added as internal reference for estimating numbers of cells.

Figure 4

Flow cytometry estimation of mitotic index in U937 cells. Cells of the histiocytic lymphoma derived cell line U937 were incubated with colcemid at 20 ng/ml for 4 h. All analyses from the same batch of cells. (a) Staining with propidium iodide ad modum Vindeløv et al. (17); mitotic nuclei (16%) represented by region P₂ in side scatter versus propidium iodide fluorescence dot plot. (b) Propidium iodide fluorescence histogram of the same sample as in Chart A; blue curve corresponds to region P₂ in Chart A. (c) Staining with propidium iodide after lysis with detergent Triton X‐100 and subsequent fixation in formaldehyde ad modum Larsen et al. (19); mitotic nuclei (23%) are represented by region P₂ in side scatter versus propidium iodide fluorescence dot plot. (d) After lysis with detergent Triton X‐100 and subsequent fixation in formaldehyde ad modum Larsen et al. (19) nuclei were additionally fixed in ethanol and sequentially stained with anti‐phospho‐histone H3 (pSer²⁸) (Sigma H9908), Alexa 488 conjugated secondary antibody (Invitrogen A11006) and propidium iodide ad modum Juan et al. (20, 21); mitotic nuclei represented by blue dots, corresponding to region P₂ in Chart C.

Figure 5

S phase fraction in female and male embryonic gonads relative to age in days pc. Female () and male (□) germ cells. Female () and male () somatic cells. Linear regression, 95% confidence limits.

Figure 6

SG ₂ M fraction in female and male embryonic gonads relative to age in days pc. Female () and male (□) germ cells. Female () and male () somatic cells. Linear regression, 95% confidence limits.

Figure 7

Number of germ cells and somatic cells in female embryonic gonads relative to age in days pc. Flow cytometry estimates of germ cells (•) and somatic cells (). Stereological estimates of germ cells (○) and somatic cells (△), data from Lutterodt et al. (9). Linear regression of log transformed data, 95% confidence limits.

Figure 8

Number of germ cells and somatic cells in male embryonic gonads relative to age in days pc. Flow cytometry estimates of germ cells (•) and somatic cells (). Stereological estimates of germ cells (○) and somatic cells (△), data from Mamsen et al. (10). Linear regression of log transformed data, 95% confidence limits.