BIOLOGY OF REPRODUCTION 56, 279-283 (1997) Intraoviductal Administration of Ribonucleic Acid from Estrogen-Treated Rats Mimics the Effect of Estrogen on Ovum Transport' Mariana Rios, Pedro A. Orihuela, and Horacio B. Croxatto 2 Unidad de Reproducci6n y Desarrollo, Facultad de Ciencias Biol6gicas, Pontificia Universidad Cat6lica de Chile, Santiago, Chile ABSTRACT In order to determine whether or not ovum transport accel- eration induced by estradiol (E 2 ) requires RNA and protein syn- thesis in the oviduct, inhibitors of RNA and protein synthesis were injected locally in rats treated with E 2 . We also tested whether administration of oviductal RNA from E,-treated rats could mimic the effect of E 2 on ovum transport. Rats on Day 2 of pregnancy were given a single s.c. injection of 10 ,ig E 2 and an intraoviductal (i.o.) injection of actinomycin D, ao-amanitin, or cycloheximide (Chx). In control groups, either the steroid or the inhibitor or both were replaced by the respective vehicle. RNA obtained from oviduct or ileum of E 2 -treated rats or from the oviduct of propylene glycol-treated rats was injected into the oviducts of recipient rats on Day 1 of pregnancy. Animals were autopsied 24 h later to determine the number and distri- bution of eggs in the genital tract. All three inhibitors partially blocked the E 2 -induced ovum transport acceleration, whereas administration of inhibitors alone did not affect oviductal egg recovery. Only oviductal RNA obtained from E 2 -treated rats decreased the number of oviductal eggs (active extract). To interpret this finding, the active extract was preincubated with RNase or DNase before i.o. administration. Other groups of recipient rats also treated with active extract were injected s.c. with Chx, or their uterine horns were ligated to disclose the fate of the miss- ing oviductal eggs. Active extract treated with RNase did not decrease the number of oviductal eggs; Chx blocked the effect of the active extract; and eggs missing from the oviduct were partially recovered in the uteri of ligated recipient rats. It is concluded that protein synthesis in the oviduct is re- quired for the full effect of E 2 on ovum transport and that one or more RNA species induced by E 2 in the oviduct are by them- selves able to mimic, and therefore mediate, the effect of E 2 on ovum transport. INTRODUCTION The duration of egg transport through the oviduct is reg- ulated by the ovarian hormones and by embryonic signals [1]. The effect of estrogen varies considerably across spe- cies and also within species depending on the dose and time of administration. Often the response to exogenous estra- diol is mixed in the sense that some eggs are expelled pre- maturely from the oviduct while the transport of others is retarded [2]. Estrogens regulate the physiology of target cells by controlling the production of specific RNAs and Accepted September 13, 1996. Received May 6, 1996. 'This work was supported by Rockefeller Foundation grants #91032 and 94025 #15 and the Special Programme of Research, Development and Research Training in Human Reproduction, World Health Organi- zation. M. Rios and P. Orihuela were recipients of research training fel- lowships funded by Wyeth-Ayerst, PLACIRH. 2 Correspondence: H.B. Croxatto Unidad de Reproducci6n y Desar- rollo, Facultad de Ciencias Biol6gicas, Pontificia Universidad Cat6lica de Chile, Casilla 114-D, Santiago, Chile. FAX: (56-2) 222-5515; e-mail: hbcroxat@genes.bio.puc.cl proteins [3]. We assumed that estradiol-17 (E 2 ) stimula- tion of RNA and protein synthesis in the oviduct is an in- termediate event essential for bringing about all subsequent changes required to accelerate or retard ovum transport, and that the different responses of egg transport to E 2 that have been described [4] reflect expression and repression of dif- ferent genes in each condition. To test the latter assumption, gene expression in the oviduct must be assessed in species and conditions in which either accelerated or retarded trans- port is a predictable response. We chose to start with the rat because egg transport in the rat features a single, pre- dictable, and well-defined response to estrogen. In this spe- cies a single injection of E 2 on Day 1, 2, or 3 of pregnancy accelerates oviductal transport of ova in a dose-related manner with a latency period of 11 h [5, 6]. In order to confirm the necessity of RNA and protein synthesis in the oviduct for this response, appropriate inhibitors were in- jected locally in the oviduct to determine their effects on E2-induced ovum transport acceleration. In addition, RNA synthesized in response to E 2 was also injected into recip- ient rat oviducts to assess whether or not it would mimic the effect of E 2 on ovum transport. The idea that the administered RNA would enter from the lumen into oviductal cells was a priori surrounded by skepticism because of the known instability of mRNA in extracellular environments due to strong and ubiquitous ri- bonuclease activity and the lack of a pathway for RNA to enter the cell across the plasma membrane. A literature search revealed very few reports in which this approach was claimed to work [7-9]. Moreover, recent reports indi- cate that oligonucleotides do enter cells and affect their function [10, 11]. Since the approach appeared relatively novel and had the potential to generate a powerful research tool, we decided to try it. Parts of the studies presented here were previously re- ported in abstract form by Rios et al. [12] and Orihuela et al. [13]. MATERIALS AND METHODS Animals Sprague-Dawley rats weighing 200-260 g were used. The animals were kept under controlled temperature (21- 24°C), and lights were on from 0700 to 2100 h. Water and pelleted food were supplied ad libitum. Vaginal smears were taken daily, and each female found to be in proestrus was caged overnight with fertile males. The presence of spermatozoa in the vaginal smear the next morning was taken as evidence that insemination had occurred, and this was designated Day 1 of pregnancy. Treatments Experiments 1-3 (see below) were done on Day 2 of pregnancy because actinomycin D (Act D) and cyclohexi- 279