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THE JOURNAL OF EXPERIMENTAL ZOOLOGY 276403-414 (1996)
Partial Purification and Characterization of Serum
Embryotrophic Factor Required for Early
Postimplantation Growth of Rat Embryos in Culture
MAKOTO USAMI AND YASUO OHNO
Division of Pharmacology, National Institute of Health Sciences, Kamiyoga,
Setagaya, Tokyo 158, Japan
ABSTRACT
Serum embryotrophic factor (SEF) required for the growth of cultured postimplantation rat embryos was partially purified from rat serum. Rabbit serum was used as a
basal medium for the embryo culture, and embryotrophic activity was measured as embryonic
protein increase. For partial purification of SEF, the ra t serum globulin fraction obtained by ultracentrifugation and (NH4I2SO4precipitation was fractionated by gel filtration, diethylaminoethyl
ion-exchange chromatography, and hydroxyapatite chromatography. Partially purified SEF was
characterized by stability tests and affinity chromatography. SEF was inactivated by heat, acid,
dithiothreitol reduction, or trypsin digestion. SEF bound to concanavalin A but not to heparin.
These results indicated that SEF was a n acid-labile acidic glycoprotein with disulphide bonds and
no affinity for heparin. The M , of SEF was estimated to be about 180 x lo3 by gel filtration. The
specific activity (U/g protein) was increased about 25-fold with 9.4% recovery by the partial purification, when 1 U of SEF was defined as the amount giving 50% embryonic protein increase. By
polyacrylamide gel electrophoresis, a protein most likely to be S E F was identified as a heterodimer
composed of subunits of M, 116 x lo3 and 62 x lo3 linked by disulphide bonds, and was shown to
be contained in the medium at micromolar concentrations. S E F appeared to be distinct from known
protein embryotrophic factors, growth factors, or cytokines. o 1996 Wiley-Liss, Inc.
Early postimplantation rodent embryos cultured
in rat serum grow as well as in the uterus (for
review, see New, 'go), and therefore their nutritional requirement can be investigated by examining the effects of serum factors in the embryo
culture. So far, many known serum factors have
been shown t o be effective for early postimplantation growth of rat embryos using elaborated
culture media. Glucose and vitamins, such as pantothenic acid, riboflavin, inositol, folic acid, and
niacinamide, were vital for embryos cultured in
dialyzed rat serum (Cockroft, '79, '88; Gunberg,
'76). High ,concentrations of rat transferrin ameliorated the anaemia of rat embryos cultured in
human serum (Cumberland et al., '87; Cumberland and Pratten, '89). Methionine and iron
promoted the growth of embryos cultured in dog
serum (Flynn et al., '87). Methionine and haemoglobin improved the growth of embryos cultured
in bovine serum (mug et al., '90). Methionine was
also reported to be essential for neural tube closure in embryos cultured in bovine serum (Coelho
and Klein, '90; Coelho et al., '89). Epidermal
growth factor, insulin, and transferrin support the
growth of embryos cultured in rat serum ex@ 1996 WILEY-LISS, INC.
hausted by repeated use in the embryo culture
(Pratten et al., '88). Insulin and insulin-like
growth factors (IGFs) improved the growth of embryos cultured in guinea pig serum (Travers et
al., '92).
Besides these known serum trophic factors,
there are yet unidentified embryotrophic factors
required for early postimplantation growth of rat
embryos, As shown in the studies using dialyzed
rat serum, some serum macromolecule is indispensable for embryonic growth (Gunberg, '76).
Cultures in heterologous sera, such as dog, human, monkey, and pig, supplemented with rat serum suggest the presence of some species-specific
embryotrophic factor in rat serum (Gupta and
Beck, '83; F'riscott, '83; Reti et al., '82; Steele, '85).
Electrophoretic analyses of protein changes in rat
serum as a medium after embryo culture showed
depletion of some unknown proteins of M , 125 x
lo3, 132 x lo3, and 214 x lo3 as well as a,-acid
Received April 1, 1996; revision accepted August 6, 1996.
Address reprint requests to Makoto Usami, Division of Pharmacology, National Institute of Health Sciences, 1-18-1, Kamiyoga,
Setagaya, Tokyo 158, Japan.
404
M. USAMI AND Y. OHNO
glycoprotein, a2-macroglobulin, and transferrin
(Klein et al., '78; Priscott et al., ,831, but none of
them has been isolated.
We have previously shown that a growth factor, called serum embryotrophic factor (SEF), necessary for early postimplantation growth of rat
embryos is present in the rat serum globulin fraction, using rabbit serum as a basal medium for
the embryo culture (Usami et al., '92). When cultured in rabbit serum alone, rat embryos grew
poorly. If rat serum was added, however, embryonic growth in rabbit serum was improved to an
extent as in Eagle's miinimal essential medium
containing the same amount of rat serum. Embryonic growth in rabbit serum was also improved
by the addition of the ra.t serum globulin fraction
prepared by (NH4),S04 precipitation. It was thus
indicated that SEF was present in the rat serum
globulin fraction. It wa3 also shown that rabbit
serum was virtually non-toxic t o rat embryos, but
lacked SEF. In addition, rabbit serum supplied
unidentified nutrients common with rat serum
and was easily preparable. Rabbit serum was
therefore considered t o be suitable as a culture
medium for the invesiigation of SEF. In the
present study, we partially purified SEF by further fractionation of the rat serum globulin fraction, using rabbit seruni as a basal medium for
the embryo culture. Subsequently we characterized and identified a protein most likely to be SEF.
MATERIALS AND METHODS
Embryo1 culture
Postimplantation rat embryos were cultured by
the roller bottle method as described previously
(Usami et al., '92). Embiryos at head-fold to early
somite stage were explanted from Wistar rats
(Crj:Wis, Charles River Jiapan, Kanagawa, Japan)
at day 9.5 of gestation. Tlhree embryos were placed
in a 30-ml bottle containing 4 ml of medium, and
rotated at 35 rpm, 37-38°C for 48 hours. Prior to
the examination of emhryotrophic activity, fractionated rat sera were concentrated to an appropriately fixed volume in each assay by ultrafiltration
using Diaflo membrane YM-10 (M, cut off 10 x lo3,
Amicon, Danvers, MA) unless otherwise noted,
and dialyzed using the SpectrdPore 7 dialysis
tube (M, cut off 50 x lo3, Spectrum, Houston, TX)
against Hanks' balanced salt solution (HBSS) containing 2 g/l glucose and 2 g/l NaHC03. The fractionated rat sera were added t o rabbit serum in
the proportion of 25% (dv), i.e., one part of fractionated serum and three parts of rabbit serum
were mixed, and the mixtures were used as culture
media. The protein content in cultured embryos was
determined by the method of Lowry et al. ('51).
Preparation of sera and the rat serum
globulin fraction
Rat serum was prepared by immediate centrifugation (Steele and New, '74) of blood taken from
the abdominal aorta of male Wistar rats fasted
for 18 hours. Rabbit serum was prepared by immediate centrifugation of blood taken from the auricular artery of Japanese White rabbits (Std:JW/
CSK, Japan SLC, Shizuoka, Japan). These sera
were heat-inactivated a t 56°C for 30 minutes and
supplemented with 1 g/l of glucose. Sera were filter-sterilized and stored a t -30°C until use.
The globulin fraction was prepared by (NH4I2SO4
precipitation after removal of lipids by ultracentrifugation. For ultracentrifugation, five parts of
rat serum (110 ml per run) was mixed with four
parts of 21% (WN) NaCl solution t o adjust the
specific gravity to about 1.063 g/ml. The mixture
was layered on 60% sucrose solution in a centrifugation tube to prevent the precipitation of the solute at the bottom. The tubes were placed in an
angle rotor (RP50-2, Hitachi, Tokyo, Japan) and
spun at 32,500 rpm (average centrifugal force
81,500g) at 14°C for 16 hours. From the non-lipid
lower layer, the globulin fraction was prepared by
(NH,),SO, precipitation as previously described
(Usami et al., '92).
Fractionation of the rat serum
globulin fraction
The globulin fraction (55 ml) was dialyzed
against 0.05 M Tris-HC1, 0.1 M NaC1, pH 8.0 (5
liter). The dialyzed globulin fraction, 5 ml per run,
was applied to a gel filtration column (Sephacryl
S-300, Pharmacia, Piscataway, NJ; 2.6 x 96 cm)
equilibrated with the dialysis buffer. The column
was run at a flow rate of 1mumin at 4°C. For M ,
estimation, a calibration curve for the gel filtration column was made with calibration kit proteins (Pharmacia).
The fractions with embryotrophic activity from
three or four runs of the gel filtration were diluted twofold by distilled water and loaded on a
diethylaminoethyl (DEAE) ion-exchange column
(DEAE Sephacel, Pharmacia, 2.6 x 19 cm) equilibrated with 0.025 M Tris-HC1, 0.05 M NaC1, pH
7.0. After washing with three column volumes of
the equilibration buffer, the column was run at a
flow rate of 0.5 mumin a t 4°C and eluted with a
PURIFICATION OF SERUM EMBRYOTROPHIC FACTOR
405
tridge was eluted by increasing the NaCl concentration to 2.0 M, and the bound fraction was collected. The unbound and bound fractions were
concentrated to the same volume by ultrafiltration using centriprep-30 ( M , cut off 30 x lo3,
Amicon).
The presence of sugar in SEF was examined by
concanavalin A (con A) chromatography. SEF was
applied on a con A column (ConA Sepharose,
Pharmacia, 1 x 8 cm) equilibrated with start
buffer (0.025 M Tris-HC1, 0.5 M NaC1, pH 7.0) at
a flow rate of 0.2 mum1 at 4"C, and the unbound
fi-action was collected. After washing with five column volumes of the start buffer, the column was
eluted with 0.3 M a-D-methylmannoside in the
Characterization study
same buffer, and the bound fraction was collected.
The HA fractions with embryotrophic activity The unbound and bound fractions were concenwere pooled and used as SEF. SEF was concen- trated to the same volume by ultrafiltration ustrated to about 2 mg proteidml by ultrafiltration ing centriprep-30.
before treatments. The treated SEF was dialyzed
Protein determination and
against HBSS prior to the assay of embryotrophic
electrophoretic analysis
activity.
Protein content in the fractions was determined
SEF was dialyzed against 0.025 M Tris-HC1,O.l
M NaCl, pH 7.0, prior to the testing of heat and by a dye binding method using Protein Assay (Bioacid stability. To test heat stability, SEF was Rad). HA fractions were analyzed by one-dimenheated in boiling water for 3 minutes and centri- sional sodium dodecyl sulphate polyacrylamide gel
fuged for the removal of the formed precipitate. electrophoresis (SDS-PAGE) as described by
To test acid stability, SEF was incubated in the Laemmli ('70) with 7.5% resolution gel (7 x 8 cm)
presence of 1M acetic acid (pH 2.3) at 25°C for 2 and 4% stacking gel of 0.75 mm thick. Samples
hours, and then dialyzed against the start buffer were prepared by mixing HA fractions with
sample buffer at 1:4 ratio with 2-mercaptoethanol
at 4°C.
SEF was dialyzed against 0.025 M Tris-HC1, 0.1 as a reducing agent, and were heated at 95°C for
M NaCl (pH 8.0) prior to the testing of sensitivity 5 minutes. The sample mixtures (5 pl)?which conagainst reduction and protease digestion. To test tained 1.0, 2.7, 1.0, and 0.1 pg of protein for HA
sensitivity against reduction, SEF was incubated fractions a, b, c, and d, respectively, were loaded
in the presence of 0.05 M dithiothreitol (DTT) and on each lane of 5 mm width. Electrophoresis was
0,025 M NaHCO, at 25°C for 2 hours, and then run at a constant current of 15 mA for 15 mindialyzed against the start buffer at 4°C. To test utes and then at 20 mA. The gel was stained with
sensitivity against protease digestion, SEF was silver (Silver Stain, Bio-Rad) and M , was estiincubated in the presence of 168 U/ml of insoluble mated from a calibration curve made with calitrypsin (from bovine pancreas, TPCK treated and bration kit proteins (Bio-Rad). The molar amount
attached t o DITC glass, Sigma, St. Louis, MO) at of specified bands was calculated from the follow37°C for 2 hours. The digestion was stopped by the ing data: relative band intensity determined as
addition of 1/32 volume of rabbit serum and the peak area by densitometry, total protein content
insoluble trypsin was removed by centrifugation.
in the sample, and the M , of the specified bands.
The heparin binding of SEF was examined by
The presence of disulphide bonds between subheparin chromatography. SEF was dialyzed against units was examined by two-dimensional diagonal
start buffer (0.025 M Tris-HC1, 0.2 M NaC1, pH SDS-PAGE under non-reducing conditions in the
7.0). The dialyzed SEF was applied on a heparin first dimension and reducing conditions in the seccartridge column (Econo-Pac heparin cartridge, ond dimension (Wang and Richards, '74) with the
Bio-Rad, 5 ml) equilibrated with the start buffer same gel as one-dimensional SDS-PAGE. Samples
at a flow rate of 1mVmin at 4°C and the unbound were prepared and applied as described above exfraction was collected. After washing with ten col- cept that 2-mercaptoethanol was omitted t o leave
umn volumes of the start buffer, the heparin car- disulphide bonds intact. After the run of the first
combined linear gradient of NaCl concentration
from 0.05 t o 0.5 M.
The fractions with embryotrophic activity from
two or three runs of the DEAE ion-exchange chromatography were pooled and dialyzed against
start buffer for hydroxyapatite (HA) chromatography, 0.05 M potassium phosphate, pH 6.9. The
dialyzed fraction was loaded on a HA column (BioGel HTP, Bio-Rad, Richmond, CA; 1.5 x 15 cm).
After washing with three column volumes of the
start buffer, the column was run at a flow rate of
0.5 or 1 mVmin at 4°C and eluted with a linear
gradient of the potassium phosphate concentration from 0.01 to 0.2 M.
M. USAMI AND Y.OHNO
406
dimension, the sample lane was excised in 1.2 mm
width from the gel, and was laid on the gel for
the second dimension. The second dimension was
run after the equilibration of the gel lane with
the sample buffer containing 2-mercaptoethanol
for 10 minutes to cleave disulphide bonds. Electrophoresis was r u n and the gel was stained
with silver i n the same way as one-dimensional
SDS-PAGE.
rat serum (% of DRS) in each run of assay to reduce the interassay variation, which was calculated as follows:
(FracEmbPro- HbssEmbPro)
Embryonic protein =
x 100,
increase (% of DRS) (DrsEmbPro- HbssEmbPro)
where FracEmbPro is protein content in the embryo cultured in rabbit serum with the addition of
fractionated rat serum of which embryotrophic acRESULTS
tivity is to be determined, HbssEmbPro is protein
Assay of embryotrophic activity
content in the embryos concurrently cultured in
For a n assay of embr!yotrophic activity at each rabbit serum with the addition of the carrier solupurification step, a quantitative and sensitive tion (HBSS) alone, and DrsEmbPro is protein conmeasure of the growth of cultured embryos had tent in the embryos concurrently cultured in rabbit
to be determined. Since the embryonic protein con- serum with the addition of dialyzed rat serum.
tent was considered most suitable among the six
Embryotrophic activity of gel
growth parameters that we had employed previfiltration fractions
ously, i.e., presence of heartbeat and flexion, yolk
The globulin fraction of rat serum showed five
sac diameter, crown-rump length, somite number,
and protein content (Usami et al., '921, we exam- major peaks when fractionated by gel filtration.
ined its relationship with the amount of SEF, in At the initial rough division into five fractions,
this case, dialyzed rat gerum expressed as pro- strong embryotrophic activity was observed in the
tein content. As a resull,, there was a clear dose- fractions 3 and 4 (Fig. 2A). When these two acresponse relationship between the protein content tive fractions were further divided into eight fracin cultured embryos and the amount of dialyzed tions, embryotrophic activity was observed with
rat serum in the 25% additives (Fig. 1);in this a peak around M , 180 x lo3 as estimated from
case 1ml of dialyzed serum, equal t o 25% in 4 ml the calibration curve made with calibration kit
of the medium, containled 68 mg of protein. On proteins (Fig. 2B).
the basis of this finding, embryonic protein inEmbryotrophic activity of DEAE fractions
crease compared to those by HBSS was used as a
The gel filtration fractions d through g were
measure for embryotrophic activity in this assay
pooled
and fractionated by DEAE ion-exchange
system. In practice, embryonic protein increase
chromatography.
When the bound fraction was
was expressed as percentage of those by dialyzed
eluted with a steep gradient of NaCl concentration and divided into four fractions, strong embryotrophic activity was observed in the fractions
2 and 3 (Fig. 3A). On further division of the fractions 2 and 3 into six fractions by elution with a
more gentle gradient of NaCl concentration,
embryotrophic activity was observed in the fractions eluted around 0.12 M NaCl (Fig. 3B). The
binding t o DEAE, a n anion exchanger, at the neutral pH indicated that SEF was acidic.
0
10
20 30 40 50 60
Dialyzed rat serum
(mgproteinl4 mllbottle)
70
Fig. 1. A plot of the amount of dialyzed rat serum in the
medium vs. embryonic protein content. Values are means for
six embryos. Error bars indicate s.e.m.
Embryotrophic activity of HA fractions
The DEAE fractions b through e were pooled
and fractionated by HA chromatography. When
the eluate was divided into four fractions according to the major peaks in the chromatogram,
strong embryotrophic activity was observed in the
fraction 3 eluted around 0.13 M PO, (Fig. 4A).
Further division of the fraction 3 showed that
embryotrophic activity was proportional to the
PURIFICATION OF SERUM EMBRYOTROPHIC FACTOR
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407
Characterization of SEF
Table 1shows the stability of SEF against heat,
acid, reduction, and protease digestion. Heat at
100°C or acid treatment with acetic acid at pH
2.3 completely inactivated SEF. Reduction with
DTT or protease digestion with trypsin also inactivated SEF although to a lesser extent. These
results indicate that SEF is an acid-labile protein
with disulphide bonds.
Table 2 shows the analysis of SEF by affinity
chromatography. For a ligand, heparin was used,
since heparin is known to bind several growth factors (Lobb et al., '86) and therefore it was expected
that their relation to SEF could be examined. On
heparin chromatography, almost all the embryotrophic activity was recovered in the unbound fraction. This means that SEF has no specific affinity
for heparin. Concanavalin A, a lectin specific for
a-D-mannopyranose or a-D-glucopyranose o r
stereochemically related sugars (Goldstein et al.,
'651, was used to examine the presence of sugar
in SEF. On con A chromatography, all the embryotrophic activity was recovered in the bound frattion. It was thus indicated that SEF had sugar.
5:
25
3
Summary of purification
The dose-response relationships at each of the
0
0
purification steps are shown in Figure 6, The slope
350
150
200
250
300
of the dose-response curve became steeper as the
Elution volume (ml)
purification
proceeded, indicating that the specific
TizPn
Gel filtration fraction
activity increased. It was noted, however, that
embryotrophic activity at the ultracentrifugation
Fig. 2. Embryotrophic activity of gel filtration fractions. and (NH4),S04precipitation was lower than that
Embryotrophic activity was expressed as embryonic protein at the start (dialyzed rat serum) and the doseincrease. Values are means for six embryos. A: Rough divi- response c u ~ e were
s
not linear exceptfor the
sion of the rat serum globulin fraction. Arrows with figures
start.
Table
3
shows
the
summary of the partial
indicate the elution volumes of calibration kit proteins with
their M , x
Vo means void volume of the column. The Purification by tentatively defining 1u of SEF as
calibration kit consists of thyroglobulin, ferritin, catalase, al- the amount that gives 50% embryonic protein indolase, albumin, and ovalbumin in descending order of M,.
crease; the amount of protein that contains 1 u
B: Further division of the fractions 3 and 4 in A. An arrow of SEF can be read from the horizontal =is at the
indicates the peak of embryotrophic activity with estimated
point of intersection of the dose response-curve and
M, x
the dotted line at 50% embryonic protein increase
in Figure 6, and its reciprocal is the specific activthird chromatographic peak (Fig. 4B),suggesting ity. The specific activities at each purification step
that SEF is a major constituent of this peak. In were calculated as units per gram of protein from
the subsequent characterization, therefore, the the results in Figure 6. From the specific activipooled HA fraction corresponding to the third chro- ties, it was indicated that SEF was purified about
25-fold. The recovery of SEF was 9.4%.
matomaphic
Desk was used.
-~
Figure 5 shows the embryos cultured in rabbit
Electrophoretic analysis of H A fractions
serum supplemented with the pooled HA fraction
as partially purified SEF. It was noted that also
The HA fractions a through d contained two
morphologically they grew well compared to the major protein bands of IW, 116 x lo3 (band A) and
HBSS control.
62 x lo3 (band B), the intensities of both of which
E
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408
M. USAMI AND Y.OHNO
.
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Elution volume (ml)
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SO
100
150
200
250
300
Elution volume (ml)
'a'b'c'd 'elf'
DEAE fraction
350
400
450
Fig. 3. Embryotrophic acti.vity of DEAE fractions. Embryotrophic activity was expressed as embryonic protein increase.
Values are means for six embryos. A Rough division of the
gel filtration fractions d through g. B: Further division of the
fractions 2 and 3 in A by more gentle gradient of NaCl concentration.
were proportional t o the embryotrophic activity
and the HA chromatog-am (Fig. 7A). In addition,
only these two protein bands showed the intensities apparently proportiional to the embryotrophic
activity at every purification step and characterization study (not shown). The bands A and B were
thus most likely to be SEF.
The molar amounts of the bands A and B in the
HA fractions a through d determined by densitometry were plotted against embryotrophic activity
in Figure 7B. Although the amounts of the bands
A and B might be somewhat inaccurate because
of possible overlapping by other faint bands, unknown sugar content, etc., the bands A and B
showed similar dose-re sponse curves, suggesting
that they were present in the same molar amount.
From this and the M , of SEF by gel filtration, it
was considered likely that the bands A and B composed heterodimeric SEF since their total M , (178
x lo3) was almost equal t o the M , of SEF by gel
filtration (180 x lo3). The dose-response curves
also show that the bands A and B were contained
in the medium at micromolar concentrations, and
that if the bands A and B compose heterodimeric
SEF, 1U of SEF as defined above was equivalent
to about 4 nmol(720 pg).
The presence of disulphide bonds between the
bands A and B was examined by the diagonal
SDS-PAGE of the HA fraction b (Fig. 7C). The
bands A and B appeared in the same shape on
the same vertical line at M , > 200 x lo3 in the
non-reducing first dimension, indicating that
they were originally linked by disulphide bonds
as a heterodimer. The inconsistency of the M , in
PURIFICATION OF SERUM EMBRYOTROPHIC FACTOR
100 I
6
I
jo
150
lbo 200
Elution volume (ml)
0.1
250
CIA fraction
, 0.06
100 I
E3
I"
0
A
160
200 300 400 500
Elution volume (ml)
'a b'c I d'
HA fraction
Fig. 4. Embryotrophic activity of HA fractions. Embryotrophic activity was expressed as embryonic protein increase.
Values are means for six embryos. A: Rough division of the
DEAE fractions b through e. B: Further division of the fractions 3 and 4 in A by more gentle gradient of PO4 concentration.
the non-reducing first dimension with the total
M , of the bands A and B is probably due to overestimation in the non-reducing first dimension;
relative mobilities of protein can be lowered in
SDS-PAGE under non-reducing conditions since
SDS tends to bind to unreduced protein in less
amount than to reduced protein (Pitt-Rivers and
Impiombato, '68).
DISCUSSION
In the present study, we partially purified and
characterized SEF in rat serum for the first time.
The results can be summarized as follows. SEF
was characterized as an acid-labile acidic glycoprotein with disulphide bonds and no affinity for
409
heparin. The M, of SEF was estimated to be about
180 x lo3 by gel filtration. Partial purification
raised the specific activity about 25-fold with 9.4%
recovery. By SDS-PAGE analysis, a protein most
likely to be SEF was identified as a heterodimer
composed of subunits of M, 116 x lo3 and 62 x
lo3 linked by disulphide bonds, and was shown
to be contained in the medium at micromolar concentrations.
The estimated M, of SEF indicates that SEF is
distinct from proteins known as embryotrophic
factors. SEF is not transferrin, the only protein
whose embryotrophic activity was directly shown
in culture (Cumberland et al., '87; Cumberland
and Pratten, '89; Pratten et al., '88). Rat transferrin, M , 68 x lo3 to 76.5 x lo3 (Charlwood, '63;
Schreiber et al., '79) is smaller than SEF, and it
was completely separated from SEF by gel filtration as pale red fractions in the fourth major peak
in the present study (not shown). SEF also seems
distinct from al-acid glycoprotein, M , 43.5 x lo3
(Nagashima et al., '80) and a2-macroglobulin, M ,
760 x lo3 (Gordon, '761, which were depleted in
rat serum used as a medium for the embryo culture (Priscott et al., '83). However, the relationship of SEF with unidentified proteins of M , 125
x lo3, 132 x lo3, and 214 x lo3 (Klein et al., '78;
Priscott et al., '83) depleted in rat serum by the
embryo culture is uncertain at present, since their
M, are near t o the estimated M , of SEF and no
other information is available.
While there has been increasing evidence that
growth factors and cytokines expressed in embryos
or uteri play important roles in postimplantation
growth (for review, see Giudice, '94; Mercola and
Stiles, '88; Tabibzadeh, '941, none of them are considered identical to SEF. Among all the known
growth factors or cytokines, only hepatocyte
growth factor (HGF) can have M , comparable to
that of SEF. The M , of HGF was initially reported
to be about 150 x lo3 by gel filtration of serum
from hepatectomized rats (Nakamura et al., '84).
Unlike SEF, however, HGF binds t o heparin and
is inactivated by incubation at 56"C, 30 minutes
(Nakamura et al., '84, '86). No affinity of SEF for
heparin also indicates that SEF is disparate from
heparin binding growth factors, a group of growth
factors that bind to heparin (Lobb et al., '86). Insulin-like growth factor-I (IGF-I), which showed
embryotrophic activity in rat embryo culture with
guinea pig serum (Pavers et al., '92), is present
in rat serum as a large complex of M , comparable
t o that of SEF. This IGF-I complex is of M , 150 x
lo3 by gel filtration, and composed of an IGF-I
M. USAMI AND Y.OHNO
410
Fig. 5. Rat embryos cultured in rabbit serum. Bars represent 1mm. A Culture witchthe addition of partially purified SEF (2.32 mg/4 ml/bottle). B: Culture with the addition
of partially purified SEF (1.16 mg/4 ml/bottle). C: Culture
with the addition of dialyzed rat serum. D: Culture with the
addition of HBSS alone.
binding component of glycoproteins of M , 29 x lo3
(IGFBP3) and 40 x lo3 associated with an acidlabile nonbinding subunit of M , 100 x lo3 (Yang
et al., '89). It is, nevertheless, unlikely that SEF
is identical t o this 1GF-I complex because the electrophoretic analysis shows no protein bands corresponding t o the components of this complex and
because it has been suggested that decreased association of IGF-I with this complex increases the
availability of IGF-I by the conceptus in pregnant
rats (Bastian e t al., '93; Davenport et al., '90;
Gargosky e t al., '90). Other growth factors or
cytokines that appeared in recent reviews (Pimentel, '94) are too small in M, compared to SEF.
The present results indicated that embryonic
protein increase was an effectual measure for
embryotrophic activity of SEF. In the past studies, embryonic growth was evaluated by combi-
TABLE 1. Stability of SEF against various treatments'
Treatment
Acid
(pH 2.3, 25"C, 2 hours)
Heat
(lOO°C, 3 minutes)
Reduction
(0.05 M D'M', 25"C, 2 hours)
Protease digestion
(168 U trypsidml, 37"C, 2: hours)
Control (% of DRS)
Treated (% of DRS)
% of control
67.1
4.8
-7.2
63.8
1.0
1.6
78.8
25.9
32.9
72.5
11.7
16.1
'Values are mean embryonic protein increase for six embryos.
PURIFICATION OF SERUM EMBRYOTROPHIC FACTOR
411
TABLE 2. Analysis of SEF by affinity chromatography'
Ligand
Unbound (% of DRS)
Bound (% of DRS)
Bound rate (%I2
Heparin
Con A
74.6
-1.2
0.4
41.8
0.5
103.0
'Values are mean embryonic protein increase for six embryos.
2Calculated by dividing the bound activity by the total of the unbound and bound activities.
nations of several parameters, such as viability,
crown-rump length, morphological feature, and
protein content. These combined parameters are
of significance for precise evaluation of the growth,
but were inadequate for the assay of SEF because
of complexity in interpreting the results; for example, crown-rump length can not be measured
without axial flexion even for the embryos with
increased protein content. The use of embryonic
protein increase as a measure made it possible to
assay embryotrophic activity quantitatively and
t o define the unit of SEF. This unit then enabled
us to calculate the specific activity. If the band A
and B protein is heterodimeric SEF, serum SEF
level can be roughly estimated to be 10 pM (1.8
mg/ml) from the specific activity in dialyzed rat
serum (37.8 U/g protein) and protein concentration in rat serum (66.4 mg/ml), because 1U of the
heterodimeric SEF is considered equivalent t o 4
nmol(720 pg). In addition to these advantages, it
should be noted that embryonic protein increase is
accompanied with morphological growth as indicated by the appearance of cultured embryos.
Besides SEF, there may be minor embryotrophic
factors that are effective for the growth of rat embryos cultured in rabbit serum. The somewhat
lowered specific activity in the globulin fraction
and non-linear dose-response curves in Figure 6
suggest that minor embryotrophic factors were
present in rat serum and lost during the purification procedures. These minor embryotrophic factors may include transferrin because it is present
in dialysis serum and has species-specificity in its
embryotrophic activity (Cumberland et al., '87;
Cumberland and Pratten, '89). Purified SEF might
be required at a higher rate for much better embryonic growth to compensate the absence of these
minor embryotrophic factors.
Rabbit serum served adequately as a basal medium for the investigation of SEF. The use of rabbit serum allowed a n assay almost specific for
SEF. Sera of other species would not have worked
for this purpose, since they can support the
growth of cultured rat embryos well without SEF.
Cultured rat embryos can grow without SEF in
human, dog, cow, or guinea pig serum, if rat
4 Start (dialyzed rat semrn)
+Ultracentrifugation and
(NH4)2S04precipitation
+Gel filtration
+DEAE ion-exchange
.s
2F
w
+
J
-
chromatography
HA chromatography
25
0
50
60
Protein content
(mg/4 ml bottle)
Fig. 6. Dose-response relationships between protein content in the fractions and embryotrophic activity at each step
of the SEF purification. Embryotrophic activity was expressed
as embryonic protein increase. Protein content represents the
amount in the 1 ml additive t o the 3 ml medium (4 ml in
total) per bottle. The purification steps are shown in order
on the right. A dotted line indicates the 50% embryonic protein increase level for the calculation of the specific activity.
Values are means for six embryos.
M. USAMI AND Y. OHNO
412
TABLE 3. Summary of SEF purification'
Purification step
Specific activity
(U/g protein)'
Purification factor (foldj3
Total activity (U4
Recovery (%I5
37.8
1
251
100
0.73
2.86
73.7
70.8
Start (dialyzed rat serum)
Ultracentrifugation and
(NH4j2S04precipitation
Gel filtration
DEAE ion-exchange
chromatography
HA chromatography
27.5
108
451
952
11.9
40.2
25.2
23.5
29.4
28.2
16.0
9.4
'Values are from a representative run of purification.
'Obtained from the results in Figire 6 by defining 1U of SEF as the amount giving 50% embryonic protein increase.
3Calculated by dividing the specific activity in each step by that of the start.
4Calculated by multiplying the specific activity by total protein content in each step.
'Calculated by dividing the total iictivity in each step by that of the start.
HA fraction
A
a
b
c
iB
d
-3
MrXlO
200
+
116 +
97.4
CBandA
+
66.2
+
45.0
+
+Band B
0
2
4
6
Protein content
(nmol/4 ml/bottle)
8
First dimension (non-reducing)
-3
C
Mrx10
b
116
66.2
HA
fraction b
-3
Mrx10
200
+
116 -b
4BandA
+
4 Band B
97.4
66.2
+
4
Heterodimer
Fig. 7. Electrophoretic analysis of the HA fractions. A
One-dimensional SDS-PAGE of the HA fractions a through
d. B A plot of the molar amount of bands A and B in the HA
fractions a through d vs. embryotrophic activity. Embryotrophic activity was expressed as embryonic protein increase.
C: Two-dimensional diagonal SDS-PAGE of the HA fraction
b. The gel shows the run of the first dimension horizontally
and the second dimension vertically. The calibration kit consists of myosin, P-galactosidase, phosphorylase b, bovine serum albumin, and ovalbumin in descending order of M,.
PURIFICATION OF SERUM EMBRYOTROPHIC FACTOR
413
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ACKNOWLEDGMENTS
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growth and physiology of mammals.
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