Developmental Programming: Impact of Prenatal Bisphenol-A on Estradiol Negative Feedback in Sheep.

Abstract

Endocrine disruptors are hormonally active substances that impact the trajectory of the developing fetus/offspring. An NHANES study found that 99% of humans studied had measurable urinary levels of bisphenol-A (BPA), a chemical with steroidogenic potential used in the plastic industry. Developmental exposure to BPA leads to detrimental effects at several levels including the reproductive axis. In sheep, prenatal exposure to BPA dampens ovulatory LH surges and induces LH hypersecretion. This study tested the hypothesis that LH hypersecretion in prenatal BPA-treated females is the consequence of reduced sensitivity to estrogen negative feedback. A dose response study was conducted involving 4 groups: control (C; n=7), BPA 0.05 mg/kg/day (B1; n=8), 0.5 mg/kg/day (B2; n=8), and 5 mg/kg/day (B3; n=7). Pregnant sheep were treated daily with BPA (in corn oil, s.c.) from days 30 to 90 of gestation. Controls received corn oil. Sensitivity to estrogen negative feedback was tested in all female offspring at ~12 weeks of age. All animals were administered a GnRH antagonist (acycline, 10 μg/kg, s.c.) every 12 h for 72 h to arrest follicular growth and suppress endogenous estradiol production. Eighty hours after cessation of acyline treatment, animals were implanted with a 3 mm Silastic implant containing estradiol (produces prepubertal levels of estradiol). Blood samples were collected at 20 min intervals for 6 h before, at the end of, and 72 h after cessation of GnRH antagonist treatment, and 67 h after insertion of estradiol implant for measurement of LH. LH pulses were detected by cluster algorithm. To test negative feedback effects of estradiol on LH pulse dynamics (frequency, amplitude, and area under the pulse), a paired t-test was used for all LH pulse variables to compare both pre- and post- estradiol periods. To assess BPA dose response relationship, the delta change between pre- and post- estradiol periods was subjected to regression analyses. GnRH antagonist treatment ablated LH pulsatility in all females. Cessation of GnRH antagonist treatment induced high frequency LH pulses in all groups. In controls, estradiol treatment suppressed LH pulse frequency 61.7±10.3% (P<0.01) and increased LH pulse amplitude and area under the pulse 152.2±65.2% (P<0.01) and 440.0±153.8% (P<0.05), respectively. Estradiol treatment also suppressed LH pulse frequency (B1: 45.2±10.1, B2: 48.3±7.2, B3: 66.1±8.8%) and increased LH pulse amplitude (B1: 200.4±34.9, B2: 157.1±38.7, B3: 302.2±101.9%), and area under the pulse (B1: 470.3±60.1, B2: 280.4±64.9, B3: 808.9±232.7%) in all 3 BPA-treated groups (P<0.01 for all doses and variables). The high degree of variability (P<0.05, homogeneity test) found within each BPA dose group, in response parameters, resulted in lack of BPA effect on LH pulse frequency and amplitude. Regression analysis uncovered a significant, but subtle positive linear correlation between BPA dose and area under the pulse (adjusted r2=0.140, P<0.05). In summary, reduced sensitivity to estradiol negative feedback, manifested as increased LH released per pulse, may be a contributing factor to the LH hypersecretion seen in these animals. The high variability in response parameters indicate that the organizational program involved in establishing sensitivity to steroid feedback is the result of the interplay between individual genetic susceptibility and developmental insults. Supported by NIH ES016541.