Activation of the aryl hydrocarbon receptor by a component of cigarette smoke reduces germ cell proliferation in the human fetal ovary

Abstract

Fetal life is a critical time for female fertility, when germ cells complete proliferation, initiate meiosis and ultimately form the lifetime stock of primordial follicles. Female fertility may be reduced by in utero exposure to cigarette smoke, which contains ligands for the aryl hydrocarbon receptor (AhR). The AhR is a critical regulator of ovarian germ cell survival in mice; thus activation of this receptor in the ovaries of fetuses exposed to maternal cigarette smoke in utero may provide a mechanism by which female fertility is reduced in later life. We have therefore investigated AhR expression in the human fetal ovary, and examined the effects of an AhR ligand present in cigarette smoke, on germ cells in human fetal ovaries cultured in vitro. The results showed that AHR mRNA expression increased 2-fold between first and late second trimester (P = 0.008). AhR protein was confined to germ cells at all gestations, but varied from expression in most germ cells during the first trimester, to only patchy expression by clusters of germ cells at later gestations. Culture of human fetal ovaries with the AhR ligand 9,10-dimethyl-1,2-benzanthracene-3,4-dihydrodiol (DMBA-DHD; a component of cigarette smoke) did not affect germ cell number in vitro, but significantly reduced the proportion of proliferating germ cells by 29% (as assessed by phospho-histone H3 staining (P = 0.04)). Germ cell apoptosis was not significantly affected. These results reveal that germ cells in the human fetal ovary express AhR from the proliferative stage of development through entry into meiosis and beyond, and demonstrate that AhR ligands found in cigarette smoke have the capacity to impair human fetal ovarian germ cell proliferation.

Keywords: fertility; germ cell; oogenesis; ovary; smoking.

Figure 1

Expression of the aryl hydrocarbon receptor AHR (A) increases with gestation (P = 0.008), but ARNT (aryl hydrocarbon translocator, an AhR co-factor) (B) was unchanged (n = 5–6 ovaries per group).

Figure 2

In the first trimester (A, 7 weeks of gestation), AhR was expressed by all germ cells (arrows) with no expression in somatic cells. At later gestations (B, 19 weeks and C, 18 weeks), AhR expression remained confined to germ cells in clusters, predominantly but not exclusively localized to the more peripheral regions of the ovary (arrows). AhR expression was low/absent in primordial follicles (D, 19 weeks). All scale bars, 20 μm.

Figure 3

In vitro exposure of human fetal ovaries (8–9 weeks of gestation) to an AhR ligand reduces germ cell proliferation. Representative images of human fetal ovaries cultured for 7 days and immunostained for phosphorylated histone H3 (A and B) and cleaved caspase 3 (C and D) indicating mitotic proliferation and apoptosis, respectively (arrows indicate immunostained cells in (A) and (C)). Exposure of first trimester fetal ovaries to the AhR ligand DMBA-DHD (1 µM) did not affect germ cell number (E), but significantly reduced human fetal ovarian germ cell proliferation relative to vehicle (DMSO) controls (F; quantified by detection of phospho-H3). Germ cell apoptosis (assessed by caspase 3 immunostaining) was not affected by DMBA-DHD treatment (G). Data are mean ± SEM of 4 independent experiments.