Effects of Neonatal Oral Exposure to Ethynylestradiol (EE) on the Puberty of Female Rat.

Abstract

[Objectives] Perinatal exposure to estrogenic compounds masculinizes the brain in female neonatal rodents and loses cyclic revolution of estrus after puberty. We have shown that oral daily administration with EE to neonatal female rats causes persistent vaginal estrus at a younger age depending on doses of EE. Although the treatment at dose levels up to 2 μg/kg BW did not affect the timing of vaginal opening (VO), it retarded the first ovulation. In order to determine effects of the treatment on the puberty, functional development of the hypothalamus/pituitary/gonadal axis towards puberty was examined by administrating exogenous gonadotropins to EE-treated immature animals. [Materials & Methods] Neonatal pups were obtained by spontaneous delivery from pregnant Sprague-Dawley rats [Crl:CD(SD)IGS]. The day when delivery was confirmed was designated as postnatal day (PND) 0 of the pups. The pups were collected and assigned to treatment groups on PND 1. The pups were identified using tattoos and placed under the same dams at eight pups per dam. If the number of treated pups was less than eight, untreated male pups were included to adjust the litter size to eight. From PND 1 to 5, the pups were given 10 mL/kg BW of corn oil (vehicle) or 2 μg/kg BW of EE orally using intubation tube, and were weaned on PND 21. Around 09:30 of PND 23, both groups of the animals were given 5 IU eCG subcutaneously, and were euthanized under sodium pentobarbital anesthesia on PND 26 to observe ovulation. Some of the animals in the both groups were given 10 IU hCG 56 hours after the eCG injection as an exogenous stimulus for ovulation. Animals treated either with corn oil or EE during the neonatal period were also necropsied on PND 23 or 26 without gonadotropins treatment. At the necropsy, the number of oocytes shed and weights of ovaries and uterus were determined. Ovarian histology was confirmed for representative cases. [Results & Discussion] Single subcutaneous injection of 5 IU eCG on PND 23 failed to induce ovulation in the EE-treated animals, while all of animals in the vehicle-treated animals ovulated by the treatment. Additional ovulatory stimulus of hCG to eCG induced ovulation to 40% of the EE-treated group with smaller number of oocytes shed than that in the vehicle-treated group. Weights of the ovaries and uterus in the EE-treated group were smaller than the vehicle-treated group at any timing of the necropsy. Ovarian histology was tended to show retardation of follicular development in the EE-treated animals. From these results, it is concluded that neonatal oral treatment with 2 μg/kg BW/day of EE could retard puberty exerting its effects probably on both the hypothalamus/pituitary and the ovary. [Acknowledgement] This study was partly supported by a grant-in-aid for research on risk of the origin of chemical compounds from the Minister of Health, Labor and Welfare (H23-Kagaku-Ippan-003).