(97) Oral Exposure to PFNA Interferes with Gonadotropin-Dependent Follicle Development and Ovulation in Mice

Antonella R. R. Caceres^1,2,3, Jiyang Zhang^1,2,3, Tingjie Zhan^1,2,3, Brian Buckley^2,3, Grace Guo¹, Nataki C. Douglas^4,5, Qiang Zhang⁶, and Shuo Xiao^1,2,3.

¹Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854, USA.

²Environmental and Occupational Health Sciences Institute (EOHSI), Rutgers University, Piscataway, NJ 08854, USA.

³Center for Environmental Exposures and Disease, Rutgers University, Piscataway, NJ 08854, USA.

⁴Department of Obstetrics, Gynecology and Reproductive Health, New Jersey Medical School (NJMS), Rutgers University, Newark, NJ 07103, USA.

⁵Center for Immunity and Inflammation, Rutgers Biomedical and Health Sciences (RBHS), Newark, NJ 07103, USA.

⁶ Gangarosa Department of Environmental Health, Rollins School of Public Health, Emory University, Atlanta GA 30322, USA.

Abstract Text:

The per- and polyfluoroalkyl substances (PFAS) are widely used in consumer and industrial products. PFAS are highly resistant to environmental degradation and metabolism after absorption, earning the name “Forever Chemicals”. Humans can be exposed to high levels of PFAS through drinking water in areas near the contaminated sites. Epidemiological evidence revealed associations between PFAS and female reproductive disorders, including irregular menstrual cycles, infertility, and premature ovarian failure, but the underlying mechanisms remain elusive. Compared to legacy PFAS (e.g., PFOS and PFOA), perfluorononanoic acid (PFNA), a newer long-chain PFAS, has similar or even higher contamination levels in multiple public drinking water supplies, including New Jersey where several authors in this study reside. Herein, we used an in vivo mouse model to investigate the ovarian impacts of human relevant exposure levels of PFNA through daily drinking water.

Seven-week-old CD-1 female mice were exposed to PFNA through drinking water at a concentration range of 0.01, 0.06, 0.4, and 4 μg/mL for 2 months, mimicking continuous oral exposure to low, moderate, and high levels of oral exposure to PFNA in humans.

Vaginal smears were conducted during the last 2 weeks of exposure to assess estrous cyclicity. Mice were euthanized on proestrus around the end of the 2-month exposure period. Blood sera and one ovary from each mouse were collected to measure PFNA using LC/MS. Serum concentration of major sex hormones were analyzed by ELISA. The other ovary from each mouse was used for isolating late-staged antral follicles for RT-qPCR to examine key genes related to follicle development, steroidogenesis, and peroxisome proliferator-activated receptor (PPAR) signaling, which has been proposed as a possible molecular target of PFAS in other organ systems. In addition, several mice treated with vehicle or 4 μg/mL PFNA were administrated gonadotropins to induce superovulation and then mated with non-exposed male mice to examine ovulation and early embryo development.

The results of LC/MS confirmed the accumulation of PFNA in both circulation and the ovary, in a dose-dependent manner. The vaginal smear results showed that, compared to the control, mice treated with PFNA had a tendency to exhibit longer diestrus and shorter proestrus. ELISA data revealed that mice from various groups had comparable serum concentrations of testosterone and progesterone, but PFNA-treated mice had decreased concentrations of estradiol although the differences were not statistically significant.

The results of RT-qPCR using isolated late-stage antral follicles revealed that PFNA reduced the expression of genes related to follicle development and steroidogenesis, including Star, Hsd3b1, and Pappa in a dose-dependent manner. Moreover, several PPAR target genes, such as Cyp27a1, Gys2, Hmgcs2, Upc1, and Cidea, were significantly increased in antral follicles from PFNA-treated mice. The superovulation and mating results showed that mice treated with 4 μg/mL PFNA in drinking water had significantly fewer eggs recovered from the oviducts on embryonic day 0.5 (E0.5) and impaired embryo development on E3.5. 

In summary, our study demonstrates that at environmentally high, but still human relevant exposure levels, oral exposure to PFNA via drinking water interferes with mouse estrous cyclicity, reduces the expression of genes related to follicle development and steroidogenesis, and disrupts ovulation and early embryo development. The induction of PPAR target genes in developing follicles suggests that PFNA acts as a PPAR agonist to perturb folliculogenesis and oogenesis.
This work is supported by NIH R01ES032144, NIH R01ES035766, NIH P30ES005022, and DOD HT9425-23-1-0809.