Animals

Two populations of animals were evaluated in this study. Population 1: CD-1 mice were ordered from a commercial supplier (Charles River Laboratories, Raleigh, NC) in adulthood (males, approximately 8 weeks of age) or at postnatal day (PND) 22 (females, prepubertal) or PND32 (females, pubertal). Upon arrival, these animals (referred to in the text as CRL animals) were group housed (typically 4 animals per cage) in polysulfone cages. Animals were provided a low phytoestrogen feed (Harlan Teklad 2018) which has previously been shown to have estrogenic activity in the low femtomolar range [32] and tap water in glass bottles. CRL animals were housed under these conditions for 2 to 3 weeks (adult males), 2 days (prepubertal females) or 3 days (pubertal females). The males were also used as breeders for other experiments.

Timed pregnant female CD-1 mice (pregnancy day 4–5) were ordered from Charles River Laboratories and housed according to the details provided above for Population 1. These dams were allowed to deliver naturally, and their litters (the F1 generation) were culled to 10 pups on PND1. Litters were weaned on PND21, the female F1 offspring were group housed with up to 2 littermates and raised to adulthood (approx. week 8). One F1 female from each litter was mated to a control male (ordered from Charles River Laboratories), and these females were also allowed to deliver naturally. Litters were culled and weaned in the same manner as the previous generation. The F2 offspring were continuously housed under controlled conditions until they reached the same ages as in Population 1. These animals will be referred to throughout the text as F2 animals.

All animals were maintained in temperature and light controlled (12 h light, 12 h dark, lights on at 0800 h) conditions at the University of Massachusetts, Amherst Central Animal Facility. All experimental procedures were approved by the University of Massachusetts Institutional Animal Care and Use Committee (protocol 2014–0055).