Adolescent exposure to a mixture of per- and polyfluoroalkyl substances (PFAS) depletes the ovarian reserve, increases ovarian fibrosis, and alters the Hippo pathway in adult female mice

Abstract

Per- and polyfluoroalkyl substances (PFAS) are synthetic chemicals known for their environmental persistence and resistance to biodegradation. This study investigated the impact of adolescent exposure to a PFAS mixture on adult ovarian function. Female CD-1 mice were orally exposed to vehicle control or a PFAS mixture (comprised of perfluorooctanoic acid, perfluorooctanesulfonic acid, undecafluoro-2-methyl-3-oxahexanoic acid, and perfluorobutanesulfonic acid) for 15 d. After a 42-d recovery period, reproductive hormones, ovarian fibrosis, and ovarian gene and protein expression were analyzed using ELISA, Picrosirius red staining, qPCR, and immunoblotting, respectively. Results revealed that PFAS exposure did not affect adult body or organ weight, although ovarian weight slightly decreased. PFAS-exposed mice exhibited a disturbed estrous cycle, with less time spent in proestrus than control mice. Follicle counting indicated a reduction in primordial and primary follicles. Serum analysis revealed no changes in steroid hormones, follicle-stimulating hormone, or anti-Müllerian hormone, but a significant increase in luteinizing hormone was observed in PFAS-treated mice. Ovaries collected from PFAS-treated mice had increased mRNA transcripts for steroidogenic enzymes and fatty acid synthesis-related genes. PFAS exposure also increased collagen content in the ovary. Additionally, serum tumor necrosis factor-α levels were higher in PFAS-treated mice. Finally, transcripts and protein abundance for Hippo pathway components were upregulated in the ovaries of the PFAS-treated mice. Overall, these findings suggest that adolescent exposure to PFAS can disrupt ovarian function in adulthood.

Keywords: PFAS; fibrosis; mixtures; ovary; steroidogenesis.

Fig. 1.

Effects of PFAS mixture on body and organ weight. (a) Body weight gain was monitored weekly. After euthanasia, (b) ovary; (c) uterus; (d) spleen; (e) liver; (f) kidney; (g) heart weights were collected. Bars represent the means±SEM; n = 8 per treatment; #=P ≤ 0.07.

Fig. 2.

Impact of PFAS mixture on estrous cyclicity and follicle number. (a) The percentage of time spent in proestrus, estrus, and metestrus/diestrus phase of the estrous cycle was determined by vaginal cytology. (b) Ovarian follicle numbers were classified and counted after exposure to vehicle control or PFAS mixture. Bars represent the means±SEM; n = 8 per treatment; #=P ≤ 0.07; *P ≤ 0.05; ***P ≤ 0.001; ****P ≤ 0.0001.

Fig. 3.

Effect of PFAS mixture on proliferation and regulators of apoptosis in the ovary. Immunofluorescence staining of the proliferation marker KI67 in ovaries from mice exposed to (a) vehicle control; (b) PFAS mixture. Immunofluorescence staining of apoptosis marker cleaved caspase 3 (CASP3) in ovaries from mice exposed to (c) vehicle control; (d) PFAS mixture. Quantification of whole ovary fluorescence intensity for (e) KI67; (f) CASP3; scale bar = 200 µm. Bars represent mean fluorescent intensity±SEM; n = 8 per ovaries per treatment; 2 sections per ovary. Ovarian mRNA levels of (g) Bax; (h) Bcl2; (i) Ki67; (j) Ccnd2 were measured by quantitative real-time PCR. Results are presented as relative fold-change means±SEM; n = 8 per treatment.

Fig. 4.

Impact of PFAS mixture on gonadotropin and sex steroid hormones. Following exposure to vehicle control or PFAS mixture, sera were collected from mice and subjected to hormone analysis. Sera measurements of (a) LH; (b) FSH; (c) AMH; (d) estradiol; (e) progesterone; (f) testosterone. Bars represent the means±SEM; n = 7 per treatment; **P ≤ 0.01.

Fig. 5.

Impact of PFAS mixture on ovarian steroidogenic gene expression. Ovarian mRNA transcripts for (a) Cyp11a1; (b) Cyp17a1; (c) Cyp19a1; (d) Star; (e) Hsd3b1; (f) Hsd17b1 were measured by quantitative real-time PCR. Results are presented as relative fold-change means±SEM; n = 8 per treatment. #=P ≤ 0.07; **P ≤ 0.01; ***P ≤ 0.001.

Fig. 6.

Effect of PFAS mixture on ovarian expression of proteins involved in steroidogenesis. (a) Representative western blot of enzymes associated with steroid synthesis in vehicle control and PFAS mixture-treated mice. Quantification of protein abundance for (b) CYP11A1; (c) CYP17A1; (d) CYP19A1; (e) STAR; (f) HSD3B1. Results are presented as relative fold-change means±SEM; n = 8 per treatment; *P ≤ 0.05.

Fig. 7.

Impact of PFAS on genes associated with cholesterol and fatty acid synthesis in the ovary. Ovarian mRNA transcripts for (a) Hmgcr; (b) Scarb1; (c) Srebp1 were measured by quantitative real-time PCR. Results are presented as relative fold-change means±SEM; n = 8 per treatment. *P ≤ 0.05; **P ≤ 0.01.

Fig. 8.

Effect of PFAS mixture on ovarian fibrosis. Representative PSR-stained ovarian sections from (a) vehicle control and (b) PFAS mixture-treated mice. Representative processed color threshold images of PSR-stained ovarian sections to quantify fibrosis from (c) vehicle control and (d) PFAS mixture-treated mice. (e) Quantification of whole ovary PSR-staining. Bars represent the means of the percent area±SEM; n = 8 per ovaries per treatment; 2 sections per ovary. Ovarian mRNA transcripts for (f) Col1a1; (g) Col4a1 were measured by quantitative real-time PCR. Results are presented as relative fold-change means±SEM; n = 8 per treatment. *P ≤ 0.05; **P ≤ 0.01.

Fig. 9.

Impact of PFAS mixture on gene expression of Hippo pathway components. Ovarian mRNA transcripts for (a) Mst1; (b) Mst2; (c) Lats1; (d) Lats2; (e) Yap1; (f) Taz; (g) Cyr61; (h) Ctgf were measured by quantitative real-time PCR. Results are presented as relative fold-change means±SEM; n = 8 per treatment. *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001.

Fig. 10.

Effect of PFAS on Hippo pathway core kinase protein abundance. (a) Representative western blot of Hippo pathway kinases in vehicle control and PFAS mixture-treated mice. Quantification of protein abundance for (b) pLATS1/LATS1 protein ratio; (c) total LATS1; (d) pYAP1/YAP1 protein ratio; (e) total YAP1; (f) CTGF. Results are presented as relative fold-change means±SEM; n = 8 per treatment; #=P ≤ 0.07; **P ≤ 0.01; ***P ≤ 0.001.