Sodium Fluoride (NaF) Releases the Two-Cell Block in Mouse Embryos

8 Pages·1993·2.8 MB·English

by  Masahisa Nakamura

ZOOLOGICAL SCIENCE 10: 629-636 (1993) 1993 Zoological SocietyofJapan
Sodium Fluoride (NaF) Releases the Two-Cell Block
in Mouse Embryos
Masahisa Nakamura1-2 Shoichi Okinaga1 Takuro Kobayashi1
, ,
and Keiji Aoki3
1Department ofObstetrics and Gynecology, School ofMedicine,
Teikyo University, Tokyo 173 and Department ofBiology,
School ofEducation, Waseda University,
Tokyo 169-50, Japan
—
ABSTRACT Thisstudywasundertakentoelucidatethemechanismunderlyingtheinabilityofmouse
embryostodevelop in culture past the 2-cell stage (2-cellblock). IncompleteTLP-PVA medium, the
rate ofdevelopment ofnon-inbred (ICR) mouse 2-cell embryos to 3- and 4-cell stages (^3 cells) was
only 11%. However,sodiumfluoride (NaF, aninhibitorofenolaseinglycolysis)significantlyimproved
therateoffurtherdevelopmentof2-cellembryosinadose-fashionmanner. NaFat5mMallowed54%
of2-cell embryos to develop to Sg3 cells, although NaFat concentrationsgreaterthan 7.5mMstunted
theirdevelopment. The removal ofglucose and phosphate from theTLP-PVA medium improved the
rate ofdevelopment of2-cellembryosto ^3cell(42%), butthe rateofdevelopmentof2-cellembryos
to ^3 cells was enhanced (77%) when NaFat 2.5mM was added to the medium without glucose and
pa[6lh-so1os4pCeh]xagaltmueic.noseTed.h.ebueNtfafneFocttat1o4fC2.N05a2mFpMraotd2sui.cg5tniimfoiMncafnortnolym14i[Cln-h01i4b2Ci]atgenldducb[oo1s4tCeh],la1a4clCttah0toe2ugpahrnoidtduh[ca14tdCi]nolnoaceftfrafoteemctp[o1r4noCdt]uhgceltuuicpootnsaekfewraoomsf
3H]deoxy-D-glucose (anon-metabolizable analogueofglucose) bytheembryos. When theintracellu-
[
lar level ofadenosine triphosphate (ATP) duringearlydevelopmentofmouse embryoswasexamined,
itwashigherbefore ratherthan aftercleavage. In addition,theATPlevelwasmuch higherbeforethe
2nd cleavage 30hr after insemination in unblocked embryos than that in blocked ones. These results
suggestthattheflowrateofglucoseinglycolysismayregulatethedevelopmentof2-cellembryosto ^3
cells, and that the intracellular level of ATP may be critical to early phase of mouse embryo
development in vitro.
(referred as "2-cell block"). This phenomenon is
INTRODUCTION not restricted to the mouse. Cattle embryos cease
development at the 8- to 16-cell stage [26], pigs at
Mouse embryos have been used for research on 4-cells [11] and hamsters at 2-cells and 4-cells [6].
pre-implantation embryology. Successful in vitro However, thisisnottrueofallmammalianspecies.
culture offertilized mouse eggsbeyond the blasto- The blocks do not occur in embryos of the rabbit
cyst stage would provide useful information on the [15], rhesus monkey [18], and humans [22]. The
early phases of development. However, with the reasons for this difference still remains unclear.
exception of some inbred and Fl strains [12, 27], Fordevelopmentof2-cell embryosinto the blasto-
2-cell embryos from non-inbred mice do not de- cyst stage in vitro, various methods have been
velop into 4-cells in a chemically defined medium employed; determination of the optimal physi-
cochemical culture environment including energy
Accepted April 27, 10, 1993 substrates [10], low oxygen concentrations [23],
Received March 1, 1993 the removalofglucose andphosphate [24], andthe
Reprint request and 2present address: Dr. M. Naka-
addition of ethylenediamine-tetraacetic acid
mura. Laboratory for Amphibian Biology, Faculty of
Science. Hiroshima University, Higashi-Hiroshima 724, (EDTA) [1]. However, the cause(s) of the block
Japan still remains far from perfect understanding.
630 M. Nakamura, S. Okinaga et al.
Recently, we reported that approximatelya half placement of embryos into culture were carried
ofthe mouse2-cell embryosdevelopedto ^3cells out as soon as possible. After5 hrincubation, the
in vitro byeliminatingglucoseandphosphatefrom eggs were washed twice with a fresh medium
the culture medium [3], although the mechanism pre-gassedwith 5% C02in airat37°C, transferred
for this is not clearyet. Ifconsidered that glucose into 0.4ml of the conditioned medium covered
is the substrate for glycolysis while phosphate with liquid paraffin oil, and incubated at 37°C
stimulates three glycolytic enzymes hexokinase, under 5% C02 in air for certain periods of time.
phosphofructokinase, and glyceraldehyde-3-
phosphatedehydrogenase, itis assumedthatwhen Experimentalstudies
glycolysis in mouse embryos is inhibited, the Experiment1: Concentrations(0, 0.5, 1.0,2.5,5,
limitations imposed by the 2-cell block can be 7.5 and 10mM) of NaF were tested for their
circumvented. This study was undertaken to ex- effects on development of 2-cell embryos to ^3
amine whether the 2-cell block is cancelled when cells in vitro.
mM
glycolysisisinhibited, usingNaFwhich isreported EmxMperiment2: Lacking 2.8 mgMlucose and 0.35
to be an inhibitor ofenolase [20]. Here we report phosphate, but with 2.5 NaF, in the
that NaF released the 2-cell block in mouse TLP-PVA medium, EDTA (100juM), which is
embryos, and that the intra-cellular level of ATP knowntocancelthe2-cellblock [14], wastestedto
may be critical in regulating the early phase of see its effect on the rate of2-cell embryo develop-
embryo development. ment beyond the 2-cell stage.
Experiment3 InconjunctionwiththeNaFeffect
:
on the development of 2-cell embryos beyond the
MATERIALS AND METHODS
2-cell stage, production of 14COz from [1-14C]-
glucose (0.29GBq/mmol; Amersham) and [6-
Collection and culture ofembryos 14C]glucose (0.26GBq; Amersham) by 2-cell
In all experiments, random-bred ICR male and embryos was determined in the presence or ab-
female mice (10-12 weeks old) were used. ICR senceof2.5 mMNaFin amediumwithoutglucose
mice were super-ovulated with intra-perioneal in- and phosphate. For determination of 14C02 pro-
jections of 5 i.u. PMSG (Teikoku Zoki) at inter- duction, fifty 2-cell embryos/tube were collected
vals 48hr apart. At 16-17hr post-hCG, the 24hr after insemination and incubated with
females were killed by cervical dislocation, the 14C]glucose (0.6nmole/tube) for 3 hr at 37°C in
[
oviducts dissected out and the cummulus masses 0.5 mloftheTLP-PVAmediumin avesselcapped
released into a TLP-PVA medium which con- tightly with a rubber stopper. Incubation was
tained 0.35 mM phosphate, 2.8mM glucose and 4 terminatedbyinjectionof0.5 mlof50% citricacid
mg/ml bovine serum albumin (BSA) [5]. For (w/v) through the rubberstopper [21]. The 14C02
fertilization in vitro, male mice were killed 14hr liberated from 14C]glucose was trapped in KOH
[
afterthefemaleswereinjectedwithhCG, thevasa and its radioactivity was determined by a liquid
deferentiaand epididymidesdissectedout andone scintillation spectrometer (Aloka, model ISC-903)
of each placed in a 0.4ml drop of pre-gassed [21].
TLP-PVAmediumwith4mgBSA/ml. Thesperm Experiment 4: 14C]Lactate production from
[
were squeezed out gently and allowed to capaci- 14C]glucose by 2-cell embryos was determined
[
tate for2hrat37°C. At 18-19hrpost-hCG, 10[A using the method of Kusaka and Ui [17]. Fifty of
of the sperm suspension was added to each 0.4ml 2-cell embryos/tube were collected 24hr after
drop ofthe TLP-PVA medium in a Falcon 35-mm insemination, and incubated with [6-14C]glucose
dish (Falcon) togiveafinalspermconcentrationof (0.6nmole/tube) for 3hr at 37°C in 0.5ml of the
150sperm///l. Eggs and sperm were incubated medium. Incubation was stopped by the addition
together for 5 hr at 37°C under liquid paraffin oil of 0.5 ml of 60% perchloric acid to the medium.
(Squibb). To minimize damage to embryos prior Two tenths of a ml ofeach sample was taken and
to culture, oviduct dissection and collection and 1.9ml of water was added before centrifugation.
The 2-cell Block in Mice 631
The supernatant was addedwith 10mgofsemicar- 1250).
bazide (Wako) and20mgofcharcoal (Wako), and
kept for 15min at 0°C with occasional shaking Evaluation ofembryos and statistical analysis
before centrifugation. One and a half ml of the Embryos were evaluated according to stage of
resultantsupernatantwasintroducedinto0.5 mlof development by counting the number of 1-, 2-, 3-
M M
0.5 glycine solution (pH9.5) containing 0.28 and4-cell embryos after certain periods oftime of
semicarbazide, 3mg EDTA (Sigma), and 2mg culture under a stereomicroscope (Nikon, model
NAD (Boehringer Mannheim). The reaction was SMZ-1). Paired comparisons were made to deter-
started by the addition of 18units of lactic dehy- mine significance.
drogenase(BoehringerMannheim),thenallowedto
proceed for 90min at 25°C before being termin- RESULTS
ated by lowering the pH below 3 with cold HC1.
Seventy mg of charcoal was then added to the
solution andthesamplewaskeptatroomtempera- Effects ofNaF, glucoseandphosphateon develop-
ture for30minwithoccasionalshaking. Charcoal, ment of2-cell embryos into 3- and 4-cell ones
separated by centrifugation, was washed twice NaF was added to the culture medium and its
with water and eluted with a mixture of 40% effectonthedevelopmentof2-cellembryosto ^3
methanol/20% ethanol/0.8% NH4OH. After 30 cells was examined. As shown in Fig. 1, NaF
min. 4ml of the effluent was evaporated to dry- significantly cancelled the 2-cell block in a dose-
ness. The resulting residue which was then dis- fashionmanner. Therateofdevelopmentof2-cell
solved in asmall amount ofwaterwas appliedto a embryos beyond the 2 cell stage reached a max-
mM mM mM
thin layer plate of cellulose (Funakoshi), and de- imum at 5 NaF. At 2.5 and 5 NaF,
veloped with the solvent system of isopropanol/ 47% (194/411) and 54% (86/160) of 2-cell
formic acid/water (6:1:1) [17]. The radioactivity embryosdevelopedto ^3cells, respectively,while
of the spot of pyruvic semicarbazone detected
under a UV lamp was counted in a liquid scintilla-
tion counter (Aloka, model ISC-903) [21].
Experiment 5: The uptake of 3H]deoxy-D-
[
glucose by 2-cell embryos was determined. Fifty
2-cell embryos/tube were collected 24hr after
insemination, and incubated with 1 nmole/tube of
2-[2,6-3H]deoxy-D-glucose (1.63TBq/mmol;
Amersham) for 3hr at 37°C in 0.5ml of the
TLP-PVA medium with or without NaF at 2.5
mM, and without glucose and phosphate. After
incubation, the embryos were washed 5 times with
fresh medium and their radioactivity was deter-
mined [21].
Experiment 6: Determination of intra-cellular
levels of ATP was carried out. After 150-200
8 10
2-cell embryos/point were incubated at 37°C for
certain periods of time in the TLP-PVA medium, NaF (mM)
(with or without 100/M EDTA), 40/A of 9% Fig. 1. The effect ofNaF on the development of2-cell
perchloric acid were added. The sample was kept embryos beyond the 2-cell stage.
astol0ut"iConfowras30admdiend,.aTnhdetAheTnP1l6ev//elloifn t2heMemKb2rCy0os3 Frriieovduestohcrotnhacefetneprtrreia-ntgsiaeosmnsisendaotffiroNensa,hFemamenbddriyuionmscucwboeanrtteeaditnraiatnngs3f7ve°arC--
was determined using the method of Singer et al. for another 43hr. The embryo stages were then
[25] using a LKB luminometer (Pharmacia, model evaluated under stereomicroscopy.
632 M. Nakamura, S. Okinaga et al.
without NaF only 11% (11/99) of 2-cell embryos Production of 14C02 and [14C]lactate from [14C]-
did. However, NaFatconcentrationsgreaterthan glucose by 2-cell embryos
7.5 mM stunted their development. We thus used 14CC>2 and 14C]lactate production from
2.5 mM NaF for further studies. When glucose 14C]glucose was [determined to see whether gly-
[
and phosphate were omitted from the culture colysis in mouse embryos was inhibited by NaF.
medium, 42% (44/106) of 2-cell embryos went Two-cell embryos were collected 24hr after in-
through a 2nd cleavage. Development of 2-cell semination and incubated for 3 hr at 37°C in the
mM
embryos to ^3-cells in the medium containing2.5 presenceorabsenceof2.5 NaFinthemedium
mM NaF, 2.8mMglucose and0.35 mMphosphate without glucose and phosphate. As shown in Fig.
(56%) was significantly higher (P<0.05) than in 3, there was no difference (P>0.5) in 14C02
the medium without glucose and phosphate (42%) production from [l-14C]glucose between mediums
(Fig. 2). When 2.5 mM NaF was added to the with or without NaF, but there was a significant
medium without glucose and phosphate, 77% difference (P<0.01) in 14C02 production from
(142/184) of 2-cell embryos underwent a 2nd [6-14C]glucose. In addition, NaF inhibited 14C]-
[
cleavage. Thisratewasquite similartothat (80%) lactate production from [6-14C]glucose by approx-
ofthe development of2-cell embryos treated with imately 50% (P<0.01) when compared to the
lOO^MEDTA (Fig. 2). control embryos (Fig. 4).
CO
o lOO
>i
u
a
e
CD 142/184 114/142
200 -
(0
-P
o
102/181 o
o
w
50
u>< 44/106
A
B
CU
26/104
T3 lOO
CU
CUD C(O0
CU
I iH
o CuD
-p
CONT -Glu NaF NaF EDTA O
-Pi -Glu U
-Pi
Fig. 2. Effects of NaF, glucose and phosphate on the
-Glu NaF -Glu NaF
release of2-cell block.
Five hr after insemination, embryos were transfer- -Pi -Glu -Pi -Glu
red to the fresh TLP-PVA medium with orwithout -Pi -Pi
2a.n8dm1M00/g^luMcoEsDe,TA0..35EmmMbrypohosswpheartee,th2e.n5imncMubaNtaeFd [l-> 4 C]glucose [6-1 4C]glucose
for another 43hr at 37°C and the ratio of 3- and Fig. 3. 14C02 productiuon from [1-14C]- and [6-14C]-
4-cells/total cells (%) was obtained by counting 3- glucose.
and 4-cell embryos under stereomicroscopy. 14COz production from [1-14C]- and [6-14C]glucose
was determined using the method as described in
"Materials and Methods". P>0.5 and P<0.01;
mM
compaired with 2.5 NaF.
i i)
The 2-cell Block in Mice 633
—
w 300 -- |— P<0.01 "
60
o o
o o
B S
a a
a a
150
ou r^- CCDO
3 U
-oua - - H301 30 -
Ck QI
jaj) >Ii
ro X
+i O
u
a>
ro NaF -a
— i—
iu
4-1
-Pyr -Pyr o
[6-1 4 CJglucose xcu
rd
Fig.4. 14C]-Labeled lactate production from [6- -ap
[
14C]glucose by 2-cell embryos.
Production of [14C]lactate from [6-14C]glucose by 3 H]Deoxy-D-glucose
2-cell embryos was determined as described in [
•'MaterialsandMethods". P<0.01;compairedwith Fig. 5. Uptake of [3H]deoxy-D-glucose by 2-cell
the addition ofNaF. embryos.
The 3H]deoxy-D-glucose uptake by 2-cell embryos
[
was determined as described in "Materials and
Methods". P>0.5; compaired with NaF.
15 2-cell embryos (2] -lOO
embryos
o
u>i
i 10
w
Hcu
-50
o
=
Time
hrs
(
Fig. 6. Intra-cellular level ofATP during the developmental stages of'mouse embryos.
Embryoswereincubatedwithorwithout 100/AAEDTAforcertainperiodsoftimeat37°CandtheATPlevelsin
theembryosweredeterminedasdescribedin"MaterialsandMethods". Dottedlinesindicatetherateofembryos
undergoing firstorsecond cleavage pertotal embryos. Toobtain these ratios, at least 50embryoswere counted
understereomicroscopyevery 1 hrduringthedevelopmentofembryosinvitro. Time meansinseminationtime.
The number of ATP determinations is indicated in parenthesis.
634 M. Nakamura, S. Okinaga et al.
imately 80% of 2-cell embryos underwent a 2nd
[3H]Deoxy-D-gIucose uptake by 2-cell embryos cleavage. This rate was quite similarto that inthe
Since NaF inhibited the glycolytic system of development of 2-cell embryos cultured in the
mouse 2-cell embryos, we examined whether the presence of EDTA.
reducedproductionof14CC»2 and 14C]lactatefrom Inhibition of the development of mouse
[
[6-14C]glucose was due to inhibition of glucose embryos by glucose may be attributable to the
uptake by 2-cell embryos by NaF. However, NaF Crabtree effect; (a) inhibition of respiration and
mM
at 2.5 had no effect on the uptake of oxidative phosphorylation by glucose [9], and (b)
3H]deoxy-D-glucose by 2-cell embryos (P>0.5) the fact that phosphate becomes rate-limiting for
[
(Fig. 5). oxidative phosphorylation and respiration in the
presence ofglucose [16]. PreviousstudiesbyAoki
ATP determination etal. [3]showedthatinhibitionofembryodevelop-
TheATPlevelinembryoswasdeterminedwhen mentbyglucoseoccurredbothinthepresence and
they were treated with orwithout 100/uM EDTA. absence of phosphate, and that the reverse was
As shown in Fig. 6, the first and 2nd cleavages also true; embryo developmentwasinhibited both
occurred at 15-22hr and 37-48hr after insemina- in the presence and absence of phosphate when
tion, respectively. The ATP level in embryos was glucose was present. By this, an inadequate ATP
found to be higher prior to the cleavage. When productionwouldoccur. Glucoseisasubstratefor
embryos were treated with or without 100pM glycolysis, whereas, as mentioned before, phos-
EDTA, there was no difference in the ATP level phate stimulatesglycolyticenzymes. Intra-cellular
12hrbefore the firstcleavagebetweentwogroups, phosphate may thus be utilized mainlyforglycoly-
but there was a difference in the ATP level 30hr sis, with consequent disruption ofphosphorylation
before the 2nd cleavage. The ATP level in reactions in mitochondria (low production of
embryos treated with EDTA was much higher ATP). As a result, poor or blocked embryo
thanthatinuntreatedones(14.6+0.6vs 10.4±2.2 development would occur. This is supported by
fmoles/cell; n=2). thefactthatthe ATPlevelinblockedembryoswas
lowerthanthatinnormallydevelopedones. When
glucose is removed from the culture medium,
DISCUSSION
phosphatecould enhance the breakdownofstored
This study clearly showed that NaF enhanced glycogentoformglucose-6-phosphate. Whencon-
the development ofmouse 2-cell embryos into the centrations of glucose-6-phosphate are high, gly-
3- and 4-cell ones. To the best ofour knowledge, colysis would be stimulated, resulting in insuf-
this is the first report indicating that treatment of ficient ATP production and inhibition of embryo
mouse embryos with NaFsignificantly releases the development. However, when phosphate andglu-
2-cell block. We have previouslyreported thatthe cose concentrations are lowered, the glycolytic
removalofglucoseandphosphatefromtheculture systemwouldbesuppressed, resultinginhighATP
medium improved the rate of development of production in mitochondria. These explanations
mouse 2-cell embryosto ^3cells (42%) [3], which for the observed inhibitory effect of glucose and
was consistent with the results obtained in this phosphate on embryo development are consistent
study. However,developmentof2-cellembryosto with the fact that NaF, an inhibitor ofthe glycoly-
^3 cells in the medium containing NaF, glucose tic enzyme, improved the rate of development of
and phosphate was significantly more enhanced mouse 2-cell embryos to ^3 cells. The suppres-
than in a medium free of glucose and phosphate sion of glycolysis by NaF is not due to a reduced
(see Fig. 2), whereas only 11% of 2-cell embryos uptake ofglucose, since this drug had no effect on
developed to ^3 cells in the complete culture the uptake of [3H]deoxy-D-glucose.
medium without NaF. It is of greatly interest to From studies of pre-implantation mouse
note that when cultured in a medium without embryos [8], glucose is known to be an unfavor-
glucose and phosphate, but with NaF, approx- ableenergysourcefordevelopmentuntilthe 8-cell
1
The 2-celI Block in Mice 635
stage. The reason for this failure of early-stage phosphorylation of p34cdc2. Further studies must
mouse embryos to develop on glucose alone is be carried out to answer these questions. Never-
found in the block at the phospho-fructokinase theless, it is of great interest to note that the
step in glycolysis [4]. This conclusion is based on development of mouse 2-cell embryos to ^3 cells
the finding that maximum glucose-6-phosphate was significantly enhanced when glycolysis is inhi-
and fructose-6-phosphate levels are much lower bitedbyNaF. The intra-cellularlevel ofATPmay
duringtheearlystages(2-and8-cellstages)thanat be critical in regulating the early phase ofembryo
later stages (morula and blastocyst stages), sug- development in mice.
gesting that glycolysis activity in mouse embryos is
low. However, embryos used for such studies [4] ACKNOWLEDGMENTS
were developed in vivo in the reproductive tract
where the 2-cell block did not take place, and so This research was supported by a Grant-in-Aid for
were obtained by being flushed from the tract. If Scientific Research from the Ministry of Education,
Science and Culture ofJapan (03640620 to M.N.)
the glycolysis ofembryos in the reproductive tract
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