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Free Radical Biology & Medicine, Vol. 23, No. 7, p. 1073–1077, 1997Published by Elsevier Science Inc.

Printed in the USA. All rights reserved0891-5849/97 $17.00 / .00

PII S0891-5849(97)00139-1

Original Contribution

PROGRESSIVE EFFECT OF a-PHENYL-N-TERT-BUTYL NITRONE (PBN)ON RAT EMBRYO DEVELOPMENT IN VITRO

TADASHI YAMASHITA,* HIDETOSHI YAMAZAKI, † YASUHIRO KON,* TOMOMASA WATANABE,*JIRO ARIKAWA,† ICHIRO MIYOSHI, ‡ NORIYUKI KASAI, ‡ and MIKINORI KUWABARA§

*Department of Animal Disease Control and §Department of Environmental Veterinary Sciences, Graduate School of VeterinaryMedicine, †Institute for Animal Experimentation, Hokkaido University School of Medicine, Sapporo, 060, Japan and ‡Institute

for Animal Experimentation, Touhoku University School of Medicine, Sendai, 980, Japan

(Received 30 August 1996; Revised 28 April 1997; Accepted 1 May 1997)

Abstract—In the present study we demonstrated the effects of the spin-trapping agent a-phenyl-N-tert-butylnitrone(PBN) on the in vitro development of rat embryos at the early stage. In rat embryos, PBN increased the speed ofthe first cleavage and had no toxicity during pregnancy after embryo culture. These results showed that reactiveoxygen species (ROIs) that were formed by activating molecular oxygens through redox reactions regulated thespeed of development for early-stage embryos. Thus, PBN caused a decrease in the level of ROIs and toxicity andan in increase in the level of the development of rat embryos. On the other hand, PBN could not decrease the 2-cellblock in vitro nor increase the blastulation rate, in contrast to the fact that a scavenger of superoxide anions, SOD,is effective in doing so for mouse embryos. From these results it was concluded that free radicals play an importantrole in the in vitro development of rat embryos at the early stage, but play no role in the decrease of the 2-cell blockor their blastulation rate. It should be noted that PBN had no toxicity for embryonic development at the 2-cellstage. Published by Elsevier Science Inc.

Keywords—a-phenyl-tert-butyl nitrone (PBN), Rat embryos, Development, 2-cell block, In vitro culture, Freeradicals

INTRODUCTION

There is evidence that free radical-induced cellular dys-function is responsible for developmental retardationduring in vitro culture of embryos of a number of spe-cies. In particular, a high oxygen concentration in theculture medium has been shown to retard embryonicdevelopment.1–6 The maternal program is replaced byembryonic genetic control during the 2-cell stage, whenthe embryonic nuclei become fully transcriptionally ac-tive7 and the bulk of the maternal messenger RNA(mRNA) is degraded.8,9 The oxygen concentration inoviduct fluid is about one-third of that in the atmo-

Address correspondence to: Dr. Mikinori Kuwabara, Departmentof Environmental Veterinary Sciences, Graduate School of Veteri-nary Medicine, Hokkaido University, Sapporo, 060, Japan.

Present address of Tadashi Yamashita: Section on BiochemicalGenetics, Genetics and Biochemistry Branch, National Institute ofDiabetes and Digestive and Kidney Disease, National Institutes ofHealth, Bethesda, MD, 20892.

Present address of Hidetoshi Yamazaki, School of Life Science,Department of Medicine, Tottori University, Yonago, 683, Japan.

sphere.10 Noda et al. demonstrated a decrease of the 2-cell block and an increase of the blastulation ratecaused by adding superoxide dismutase (SOD), a scav-enger of superoxide anions, to mouse embryos in cul-ture medium.11 These facts suggested that oxygen rad-ical species were involved in the developmental eventsof embryos in vitro. Many reagents such as ATP, nu-cleotide monophosphate (AMP, GMP, TMP, UMP,CMP and nicotinamide adenine dinucleotide) as wellas many kinds of amino acids and vitamins in the me-dium also assist in the development of rat embryos,12

although these reagents are not required for mouse em-bryos.

a-Phenyl-N-tert-butylnitrone (PBN) is a widelyused spin-trapping agent which can trap not only ox-ygen radicals but also free radicals that are induced byCCl4 in rats.13 This reagent diminished the increase inthe amount of oxidized proteins under the cortical isch-emia/reperfusion injury to gerbil brain.14 Chronic treat-ment of PBN caused a decrease in the level of oxidized

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protein in aged gerbil brain.15 Studies concerning tissuedistribution, excretion and metabolism showed thatPBN is rapidly absorbed and distributes in many tissueswhen intraperitoneally injected into animals. Liver isthe site of metabolism and adipose tissue is the site ofstorage for PBN.16 Therefore, it is of interest to examinewhether PBN influences the early stage of developmentand gene expression in embryos.

In the present study, we investigated the effects ofPBN on embryonic development in vitro, toxicity, therelease of the 2-cell block and the subsequent increaseof the blastulation rate. The results showed that PBNaffected normal embryonic development at an earlystage, and that PBN increased the speed of the firstcleavage with no toxicity during the pregnancy afterthe transfer of 2-cell embryos. PBN was also expectedto stimulate the release of the 2-cell block in rats sinceSOD was reported to decrease the mouse 2-cell blockand to increase the blastulation rate, 11 but PBN had noeffect on the decrease of the 2-cell block or the blas-tulation rate in rat embryos.

MATERIALS AND METHODS

Animals and bleeding

Female rats, 11- to 13-week-old virgins of WKAH/Hkm Slc, were purchased from Japan SLC, Inc., To-kyo. They were superovulated with intraperitoneal in-jection of 40 IU of pregnant mare serum gonadotrophin(PMSG; Teikokuzouki, Co. Ltd., Tokyo, Japan) fol-lowed by injection with 20 IU human chorionic gonad-otropin (hCG; Teikokuzouki, Co. Ltd.) at 48 h, andwere individually housed overnight with male adults.The presence of a vaginal plug the next morning (20–24 h after hCG) was signal of the first day of preg-nancy. Animals were maintained at 23 { 37C with rel-ative humidity of 50 { 5%. Rats were fed regular lab-oratory diet and water ad lib. All experiments weredone according to the Guide for the Care and Use ofLaboratory Animals, Hokkaido University School ofMedicine.

Embryo collection

The female rats having vaginal plugs were anesthe-tized by ether and killed by cervical dislocation. Theembryos were collected from the oviducts at noon onthe first day of pregnancy into modified Krebs-Ringerbicarbonate solution (mKRB) with 4 mg/ml bovine se-rum albumin (BSA, FracV; Sigma Chemicals Co., St.Louis, MO, USA) and were treated with mKRB with0.1% hyaluronidase (Sigma Chemicals Co.) to removecumulus cells, rinsed twice with mKRB without hyalu-ronidase, and pooled in a 50 ml drop of mKRB. Twenty

to 30 embryos with normal morphology were trans-ferred into a drop of experimental medium at randomand then cultured at 377C under 5% CO2. Degenerateembryos including non-polar cells or only one male orfemale pronucleus were excluded at 0 hr.

Reagent and embryo culture medium

The embryo culture medium was mKRB supple-mented with PBN (Sigma Chemicals Co.) with 5.6 and22.6 mM. A 50 ml drop of medium was placed into eachwell of the dishes (Nunc, Denmark) and covered withparaffin oil (Sigma Chemicals Co.) . Embryos were ob-served with a microscope every 12 h.

Embryo transfer

To transfer the embryos that were cultured for 24 or48 h, pseudopregnant Sprague-Dawley rats (Japan SLCInc., Tokyo) mated with male rats whose spermiductswere cut, were used as foster mothers. After the culture,2-cell embryos with normal morphology were trans-ferred into the oviducts of pseudopregnant foster fe-males. Newborn pups were observed on day 22 aftermanipulation.

Statistical calculation

Data were expressed as mean { standard deviation,and statistical analysis was carried out using Student’st test.

RESULTS

The effective concentration of PBN to develop ratembryos

The results show (Fig. 1) that PBN increased thedevelopmental speed of rat embryos from the 1-cell tothe 2-cell stage. More than 80% of the embryos devel-oped to the 2-cell stage in 24 hrs in PBN containingmedium. Embryos cultured with PBN containing me-dium show the cleavage to 2-cell stage faster than cul-tured without PBN containing medium. After 24 hrs,the cleavage rate of the embryos cultured with PBNreached a plateau. On the other hand, the embryos cul-tured without PBN show the rate increasing. At 48 hrs,90% or more of the embryos cultured with PBN, butonly 60% of the embryos cultured without PBN de-veloped to the 2-cell stage.

Developmental ability after exposure to PBN

Table 1 shows the number of newborn pups obtainedfrom embryos that were or were not treated with PBN

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Fig. 1. Developmental rate to the 2-cell stage with or without PBN at 0, 12, 24, 36 and 48 hours. Open squares show embryoscultured without PBN (control) . Solid circles show embryos cultured with PBN at 5.6 mM. Solid squares show embryos culturedwith PBN at 22.6 mM. The numbers of embryos examined were 77 (control) , 88 (PBN 5.6 mM) and 108 (PBN 22.6 mM). Valuesare totals from four replicate experiments.

**Significantly different, p õ .01. *Significantly different, p õ .05.

Table 1. Fifteen Embryos Treated with PBN or without PBN for 24h were Transferred to Each Oviducts of Pseudopregnant Females

MediumCondition

No. ofTrials

No. ofEmbryosExamined

No. (%) ofNewborn Pups

with PBN 3 90 38 (42.2)without PBN 3 90 39 (43.3)

A foster mother had 12 to 14 new pups and raised them to over 4weeks old. The new pups showed no morphological changes andmalformations after their birth.

for 24 h. Fifteen embryos at the 2-cell stage were trans-ferred to each oviduct. About 40% of embryos becamenewborn pups. There were no difference in the numberof newborn pups between the embryos with PBN andthose without PBN. Furthermore, no morphologicalchanges and no malfunctions of newborn pups wererecognized. Table 2 shows the same examination for48-hrs incubation. The results showed that no pupswere born in either group. These data clearly provedthat PBN had no toxicity in the development of theearly-stage embryos and did not promote malformationof the fetus.

DISCUSSION

Toxicity of PBN against rat embryos

In mammals, all cells including the early stage ofembryos are at risk for injury by reactive oxygen spe-cies (ROIs) formed by activation of molecular oxygensduring redox reactions in the cells. The development ofembryos is also thought to be regulated by ROIs thatreact with proteins, lipids and nucleic acids to producereactive intermediates. These intermediates, as well asROIs themselves, can be trapped by employing spin-trapping reagents. Therefore, it is considered that trap-ping of the intermediates as well as ROIs and the sub-sequent interruption of further biochemical reactionsby the spin-trapping reagents may decrease embryotoxicity due to ROIs. In this research, we used PBN asa trapping reagent. First, we demonstrated that PBNhad no toxicity against rat early-stage embryos. Sinceearly embryos are multipotent cells, this result is par-ticularly surprising in view of the fact that PBN showedno toxicity even at relatively high concentrations, pro-viding a novel reason for PBN to be employed as a trapof ROIs in embryos.

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Table 2. Fourteen or Fifteen Embryos Treated with or without PBNfor 48 h were Transferred to Each Oviduct

of Pseudopregnant Females

MediumCondition

No. ofTrials

No. ofEmbryosExamined

No. (%) ofNewborn Pups

with PBN 4 119 0 (0)without PBN 3 90 0 (0)

No pups were born from foster mothers.

Morphological changes of rat eggs after treatmentwith PBN

A certain percent of the embryos degraded duringculture. The degraded embryos could not be transferredto the foster mother because morphological featureswith abnormal changes were correlated to abnormal de-velopment or embryo death. Thus, embryos did not de-velop to the next stage. Pronucleus-stage embryos de-velop to the next stage under the restricted control oftime in vivo and in vitro. This means that embryonicdeath occurs if the control of time is disturbed becauseof a concentration imbalance among ions, O2 and CO2

in culture medium. PBN morphologically maintainedthe normal features of embryos and increased the de-velopmental rate from the pronucleus-stage to the 2-cell stage. This result also supported the idea that PBNdecreased the embryonic toxicity due to ROIs.

Maintenance of developmental ability in the presenceof PBN

The developmental ability of the embryos is main-tained when they are cultured in medium with or with-out PBN for 24 h. After 24 h, all of the 2-cell stageembryos lost their developmental ability. Medium withPBN couldn’t keep their developmental ability after 24h. After this time, there was no difference than whencultured without PBN. However, the ratio of morpho-logical changes of embryos cultured with PBN wereless than that without PBN after 24 h. It was clearlyshown that ROIs are involved in the developmentalspeed of the early stage embryos. PBN might preventthe embryos reaching the degradated stage. It is inter-esting if the events of embryos degaradation of the re-lated with a specific ROI species.

Effects of PBN on the release of the 2-cell block

Culture for 48 hrs usually results in 4-cell embryos,but the 2-cell block can not be released in vitro in ratembryos. In some mammals, evidence has been ob-tained for an arrest of development at specific stages,the 2-cell stage in the mouse,17 4- to 8-cell stages in

human,18 2-cell stage in the hamster,19 8- to 16-cellstages in sheep and goats,20 4- to 8-cell stages in thecow,21 and 4-cell stage in the pig.22 In mouse embryos,the 2-cell block can be released by addition of antiox-idants such as SOD,11,23 suggesting that the 2-cell blockmight be a result of free radical toxicity associated withthe oxidative trauma of zygote collection and culture.Rat embryos might be more sensitive to free radicaltoxicity than mouse embryos because the antioxidantPBN was not effective to release the 2-cell block. It isnot yet clear what components release the 2-cell blockin rat embryos.

The culture of rat embryos in vitro is more difficultthan that of mouse embryos. Thus, some trials to re-lease the 2-cell block of rat embryos have been unsuc-cessfully carried out. It has been established that 1-cellembryos develop to the blastocyst stage followinghatching in mice. In this study, the 2-cell block couldnot be released by PBN. The ROIs responsible for in-hibiting the release of the rat 2-cell block were notclear. However, considering other animals such as miceand cattle, it is inferred that rat embryos are also influ-enced by ROIs that do not react with PBN.

Long-term culture in vitro with PBN

PBN could keep the embryos in good shape (non-degraded form) in vitro culture. At 24 h. when embryoshave a developmental ability to 2-cell stage, only 3%embryos show degraded form cultured with PBN inboth concentration (5.6 and 22.6 mM), but 20% of theembryos cultured without PBN exhibit the degradedform. At 48 hrs, embryos cultured with and withoutPBN show the degraded form at 8% and 35%, respec-tively (data not show). Thus, PBN decreased the rateof degraded embryos. Although the most effective con-centration of PBN for the development of embryos re-mains to be clarified, PBN certainly prevented embry-onic degradation leading to death.

Acknowledgements — The authors thank Mrs. Tsutomu Osanai,Kishio Ishikawa and Yukio Endoh of the Institute for Animal Ex-perimentation, Hokkaido University School of Medicine for technicalsuggestions and animal treatments. We also thank Ms. Fran Norflusfor correcting the manuscript. In conducting the research describedin the text, the investigators adhered to ‘‘The Guide for the Care andUse of Laboratory Animals, Hokkaido University School of Medi-cine.’’

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