Prenatal exposure to diethylstilbestrol has multigenerational effects on folliculogenesis

Abstract

Diethylstilbestrol (DES) is an estrogenic endocrine disrupting chemical (EDC) that was prescribed to millions of pregnant women worldwide, leading to increased rates of infertility in the exposed offspring. We have previously demonstrated that this reduced fertility persists for multiple generations in the mouse. However, how altered ovarian function contributes to this infertility is unknown. Therefore, this study sought to determine if DES exposure promotes two common ovarian disorders, primary ovarian insufficiency (POI) and polycystic ovary syndrome, contributing to the reduced fertility in DES offspring. Moreover, we investigated if these impacts are transgenerational. Gestating mice were exposed to 100 µg/kg DES, and ovarian morphology was observed in F1-F3 female descendants. F1 females trended towards fewer primordial and more secondary follicles and similarly, F2 females had fewer primordial and significantly more secondary follicles compared to controls. No differences in follicle proportions were observed in the F3. Moreover, DES exposure did not increase follicular cysts. These results show that DES accelerates folliculogenesis, indicative of a POI phenotype and that this is likely contributing to the reduced fertility observed in DES descendants. Moreover, this study highlights the ability of estrogenic EDCs to disrupt folliculogenesis, which may exacerbate the onset of POI in women already at risk.

Fig. 1

Body weight and AGD of young cohort. (a) DES exposure did not have any significant effect on body weight or (b) standardised AGD in the young cohort in any generation. (n = 10, 10, 5 and 11 for vehicle, F1, F2 and F3 respectively).

Fig. 2

Effect of prenatal DES exposure on follicle numbers. Ovaries were collected from PND21 control and F1-F3 DES treated females and serially sectioned at 7 μm. Every 10th section was used to count primordial, primary, secondary and antral follicles. a) The F1 generation trended towards more secondary follicles compared to the control. (b) DES significantly increased the number of secondary follicles in the F2 generation. (c) No significant difference in follicle numbers were observed in the F3 generation. (* p < .05) (n = 8, 10, 4 and 8 for vehicle, F1, F2 and F3 respectively).

Fig. 3

Effect of prenatal DES exposure on proportion of follicle types. Follicle types represented as a proportion of the total number of healthy follicles. (a) The F1 generation trended towards fewer primordial and more secondary follicles compared to the control. (b) DES significantly increased the proportion of secondary follicles in the F2 generation. (c) No significant difference in follicle proportions were observed in the F3 generation. (* p < .05) (n = 8, 10, 4 and 8 for vehicle, F1, F2 and F3 respectively).

Fig. 4

Proportion of atretic follicles in young cohort. Follicles were defined as atretic if two or more of the following features were present: degenerating oocyte, GC layer containing pyknotic nuclei, detached GC layer from the basement membrane or a broken basement membrane. DES exposure had no significant effect on the proportion of atretic follicles in any generation.

Fig. 5

IHC sections of PND 21 control ovaries. (a) and (b) are representative images of PCNA stained ovaries. (c) and (d) are representative images of Casp3 stained ovaries. (a) and (c) scale bar = 200 μm, (b) and (d) scale bar = 50 μm.

Fig. 6

GC proliferation and apoptosis was analysed via PCNA and Casp3 antibodies respectively. (a) DES exposure had no significant effect on GC proliferation across any generation. (b) DES increased the median percentage of Casp3 positive follicles in the F1 generation, however this was not significant. (PCNA, n = 5, 3, 2, 3 and Casp3, n = 5, 3, 3, 3 for vehicle, F1, F2 and F3 respectively).

Fig. 7

IF sections of PND 21 ovaries. No difference in GC (FOXL2; green) or theca cell (COUP-TFII; red) morphology was observed. (a) represents a control ovary; (b) represents a F1 DES treated ovary. Scale bar = 25 μm.

Fig. 8

Body weight and AGD of aged cohort. (a) Median body weight was increased in F1 and F2 DES females, although this increase was not significant. (b) DES exposure significantly reduced the standardised AGD in the F1, F2 and F3 generation. (* p < .05; ** p < .01) (n = 7, 4, 3, 7 respectively).

Fig. 9

Number of cystic follicles in aged cohort. A follicular cyst was defined as a fluid-filled structure lacking GCs and an oocyte and lined by one to four layers of cuboidal GCs. DES exposure had no significant effect on the number of cystic follicles.

Fig. 10

H&E stained ovarian sections representing various follicle types. (a-c) represent healthy follicle types, (d-f) represent atretic follicles. P, primordial follicles; Pr, primary follicles; S, secondary follicles; A, antral follicles; At, atretic follicles. Scale bar = 25 μm.