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Embryo growth and differentiation factors in embryonic sera of mammals
DEVELOPMENTAL
BIOLOGY
76,465-474
(1980)
Embryo Growth and Differentiation Factors in Embryonic Mammals YU-CHIH
Sera of
Hsu
Laboratory of Mammalian Development and Oncogenesis, Department of Pathobiology, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland 21205 Received August 14, 1979; accepted in revised form December 5, 1979 Mouse embryos sequentially developed in vitro were classified by stages according to several established in vivo criteria. At least three heat-sensitive macromolecules from embryonic sera were found to be successively required for mouse embryos to continuously develop from stage 6 to stage 15 in vitro. Fetal calf serum (FCS) was found to be required for in vitro development only from stage 6 to stage 11, and human placental cord serum (HCS) was found essential from stage 11 to stage 15. The factors have been tentatively designated as embryo growth and differentiation factors (EGDF). FCS was separated by molecular sieving into at least two fractions: a large fraction (EGDF-1) and a smaller fraction (EGDF-2). EGDF-1 is required for embryos to develop from stage 7 to stage 8, EGDF-2 is required from stage 8 to stage 11. EGDF-3, the factor in human cord serum, was found to be indispensable for mouse embryonic development from stage 11 to stage 15. Since blastocysts (stage 6) were able to develop to stage 15 in the medium containing HCS as the sole source of macromolecules [Hsu, Y-C., et al. (1974). J. Embryol. Exp. Morphol. 31,235-2451, it is apparent that HCS also contains EGDF-1 and EGDF-2. Embryonic development was significantly retarded in decreasing concentrations of the three EGDFs. INTRODUCTION
Mouse embryos during the preimplantastage develop adequately in KrebsRinger’s bicarbonate solution with pyruvate, lactate, or glucose as a sole energy source for cleavage. These developing embryos during the cleavage stages do not require macromolecules or exogenous nitrogen for embryonic development. As a result, at the end of the cleavage stage, the total protein content of the whole embryos is decreased by 20% (Brinster, 1967). In contrast to the preimplantation stage, mouse embryos abruptly and exponentially increase their embryonic mass immediately after implantation. It has been reported that both amino acid and macromolecules are required beyond the implantation stage (Gwatkin, 1966a; Spindle and Pedersen, 1973). These drastic metabolic changes, from dependence on stored nutrients to an exogenous source uptake, are triggered by tion
the embryonic attachment at the implantation stage, thus offering many interesting problems in the field of implantation biology. It is intriguing to study the macromolecules which regulate the rapid growth and differentiation of embryonic development. On the other hand, it has been reported previously that it is now possible to develop mouse embryos in vitro from the two-cell egg stage (stage 2 of Witschi, 1972; Theiler, 1972) to the early somite stage (stage 15 of Wits&i and stage 13 of Theiler) (Hsu et al., 1974). Subsequently, after modification and improvement of the culture method, about 50-70% of the individually cultured blastocysts (stage 6) differentiated to stage 15 if there were frequent medium changes to remove embryonic waste (Hsu, 1979). The two-cell eggs of the mouse embryos were initially cultured in a chemically defined medium up to the blastocyst stage (stage 6). Then they were transferred to a commercial
culture
medium
which
contained
465 OOlZ-1606/80/060465-10$02.00/0 Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved
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DEVELOPMENTAL BIOLOGY VOLUME76,198O
calf serum or fetal calf serum. Blastocysts continued their growth after shedding the zona pellucida, attached to culture dishes, and formed egg cylinders. Several laboratories are now able to routinely culture mouse embryos from stage 6 to stage 11 with a commercial culture medium using fetal calf serum as a sole source of macromolecules (Pienkowski et al., 1974; Sherman, 1975; McLaren and Hensleigh, 1975; Juurlink and Federoff, 1977; Wiley and Pedersen, 1977). Because human placental cord serum is not commercially available, several laboratories had attempted to culture mouse embryos in vitro to the stage of early somite (stage 15) by using commercial fetal calf serum as a sole source of serum in the culture medium. Although some degree of embryonic growth continued beyond stage 11 in the medium using fetal calf serum as a sole source of macromolecules, the structure of the mouse embryos became increasingly distorted as compared to that developed in utero. The neural plate atrophied and eventually disappeared. These results were consistent. Mouse embryos, however, resumed their normal growth in vitro, if fetal calf serum was replaced by human placental cord serum beyond stage 11 (Hsu, 1973, 1979). These in vitro mouse embryos, at any stage between stages 6 and 15, were indistinguishable from those grown in utero to corresponding stages (Hsu et al., 1974;Wiley and Pedersen, 1977; Gonda and Hsu, 1980; Libbus and Hsu, 1980). These results suggested either that fetal calf serum lacks a factor for development of mouse embryos from stages 11 to 15 which is present in human placental cord serum or that fetal calf serum contains an inhibitor(s) against the factor in human placental cord serum or both. In order to attain the long-range goal of the study of the macromolecules which regulate rapid growth and differentiation of embryonic development, this paper reports
on some of the biological assay methods used to identify the macromolecules. MATERIALS
AND
METHODS
Sera Fetal calf serum (FCS’). The FCS (from Grand Island Biological Co., New York) is heat-inactivated at 56°C for 30 min and stored at -20°C until used. Human placental cord serum (HCS). Blood is withdrawn from the umbilical cord while the placenta is still in the uterus. The vacutainer containing cord blood is placed at 4’C. Cord blood is separated from blood clots by centrifugation at 4000 rpm for 30 min as soon as possible but not later than 24 hr after being withdrawn from the umbilical cord. Hemolyzed cord serum is discarded. Cord serum is heat-inactivated at 56°C for 30 min and used on that day or stored at -60°C and used within 2 days. Serum Fractionation by Amicon Diaflo Membrane Ultrafiltration Amicon Diaflo ultrafiltration membranes (XM300, XMlOO, XM50, PM30, and PMIO) were soaked in distilled water for at least 1 hr before use to remove coated glycerol. The membranes and support were then immersed in 5% formalin for sterilization with the glossy side toward the solution. After riming three times with 30 ml of sterile distilled water, membranes were placed on the supports and prefiltered with 10 ml of sterile distilled water two times to remove residual formalin. The stirred cell (Model 52 or 402) was sterilized by autoclaving without a supporting pad. Sera were ultrafiltered under 5% COZ and 95% Nz gas pressure of 10 to 20 psi. Fifty milliliters of heat’ Abbreviations used: FCS, fetal calf serum; HCS, human placental cord serum; BM, basal medium; BSA, bevin: serum albumin; EGDF, embryo growth and differentiation factor; F > XM300, FCS ultrafiltrate fraction not permeable through XM300, XM300 > F > XMlOO, FCS ultrafiltrate fraction permeable through XM300 but not through XMlW, XMlOO > F > PM30, FCS ultrafiltrate fraction permeable through XMlOO but not through PM30.
Yu-CHIH
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Embryo Growth Factors of Mammals
inactivated fetal calf serum was ultrafiltered through the Amicon Diaflo membrane XM300 in the Model 52 stirred cell, and 45 ml of ultrafiltrate was obtained. Fifteen milliliters of medium CMRL 1066 was added to the 5 ml of retained fraction in the stirred cell (washout). The ultrafiltration was continued. This portion of ultrafiltrate was discarded. The “washout” with CMRL 1066 was repeated three times. The fetal calf serum fraction retained in membrane XM300 was designated F > XM300. The 45 ml of filtrate through XM300 was further ultrafiltered against XMlOO. The 35 ml of ultrafiltrate so obtained was designated fraction F < XMlOO. The fraction retained in the stirred cell was washed out three more times with 20 ml of medium CMRL 1066. The final portion retained in membrane XMlOO was designated XM300 > F > XMlOO. Preparation
of Blastocysts
Random bred CF 1 mice 6 weeks old were purchased from the Charles River Co., Massachusetts. Follicular growth was stimulated in the females by the injection of 5 IU of pregnant mare serum gonadotrophin (Organon, Inc., West Orange, New Jersey). Five IU of human chorionic gonadotropin was injected 48 hr later to stimulate ovulation. The female mice were then placed with males, one pair per cage. The next morning, the females were checked for the presence of a vaginal sperm plug (Day 0 of pregnancy). On the third day of pregnancy, they were killed by dislocation of the neck. All procedures in the preparation, cultivation, and observation of mouse embryos were conducted at an ambient temperature of 37°C under either a horizontal or a vertical flow hood. The uteri were removed from their mesometrium and placed in lOOmm plastic culture dishes. Using a dissecting microscope, each uterine horn was flushed with 1 ml of basal medium (BM) which consisted of medium CMRL 1066 plus 0.25% bovine serum albumin (BSA)
467
(Miles Laboratories, Inc.) Blastocysts were sucked into a capillary pipet and pooled in a 35-mm plastic culture dish containing 2 ml of basal medium; the dish was constantly flushed with 5% COZ and 95% air to keep the pH of the medium at 7.4. Ten blastocysts were distributed by capillary pipet into separate culture dishes, each of which contained 2 ml of BM. The cultures were maintained at 37°C in humidified incubators containing 5% COZ and 95% air. The concentration of CO2 was automatically regulated. Biological Assay of Embryo Growth Differentiation Factors (EGDF)
and
Since the biological activities which promote the growth and differentiation of mouse embryos have not been identified, the term “factor” will be tentatively used for convenience in this paper. Biological assay of EGDF-1. Mouse blastocysts were flushed out of the uterine horns with 1 ml of BM. A fraction of FCS was serially diluted with 2 ml of BM in 35mm Flacon tissue cultures dishes. Ten blastocysts were placed in each culture dish. After 2 days of incubation, when most of the blastocysts had shed the zona pellucida, the blastocysts were scored into three categories: (1) blastocysts floating on culture medium and not attached; (2) blastocysts slightly attached to culture dishes but trophoblasts not spread out (Fig. 1A) (both (1) and (2) are classified as stage 7); and (3) blastocysts attached and trophoblasts spread out on culture dishes (stage 8) (Fig. 1D). Biological assay of EGDF-2. About 10 blastocysts (stage 6) were initially cultured in 35-mm culture dishes which contained 2 ml of medium CMRL 1066 plus 10% fetal calf serum. After 2 days, when the blastocysts had attached to the culture dishes, the culture medium was removed and rinsed once with 2 ml of BM. Then the attached blastocysts at stage 8 were further cultured in BM plus a serial dilution of each
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FIG. 1. Embryo growth and differentiation factor (EGDF) 1. (A) Blastocysts (stage 6) were cultured in BM, which consists of medium CMRL 1066 plus 0.25% BSA. After 2 days in culture, blastocysts shed the zona pellucida and attached lightly to the surface of the culture dishes. Mural trophoblasts, however, did not spread out (stage 7). (B) After 3 days in culture. (C) After 4 days in BM, trophoblasts began to degenerate. (D) Fetal calf serum or F > XM300 was added in BM before trophoblasts degenerated, or blastocysts (stage 6) were cultured in BM for 2 days; trophoblasts spread out on culture dishes, leaving an inner cell mass on the center of the trophoblast cell sheet (stage 8). X 400.
fraction of serum ultrafiltrate or FCS. After further incubation for 2 days, the embryos were scored and classified by developmental stages as stages 8,9, 10, and 11. Biological assay of EGDF-3. Blastocysts were initially cultured in 2 ml of medium CMRL 1066 plus 10% FCS for 3 days. Then the medium was replaced by 2 ml of BM which contained different serial concentrations of HCS in different cultures. The medium in each case was renewed daily with the same concentration of HCS. Embryos
were again scored and classified at the end of 7 days’ cultivation. RESULTS
The mouse embryos which developed in vitro were classified into stages according to the in uiuo criteria of Witschi (1972), Theiler
(1972), and Hsu (1979).
EGDF-1 Mouse blastocysts were initially cultured in BM consisting of medium CMRL 1066
Yu-CHIH
Hsu
469
Embryo Growth Factors of Mammals
plus 0.25% BSA. When blastocysts (stage 6) were cultured in BM, they shed their zona pellucida and attached lightly to the culture dishes (stage 7) after 2 days of culture (Fig. 1A). The trophoectoderm, however, did not proliferate or spread out on the surface of the culture dishes. Blastocysts remained in stage 7 (Fig. 1B) for 2 or 3 days if kept in BM, and then gradually disintegrated (Fig. 1C). If BSA was replaced by FCS before the blastocysts had died, the trophoblast resumed proliferation and spread out (Fig. 1D). Fetal calf serum was fractionated into three fractions-two higher molecular weight fractions, F > XM300 and XM300 > F > XMlOO, and a lower molecular fraction, XMlOO > F > PM30-by ultrafiltration through Amicon Diaflo membranes XM300, XMlOO, and PM30. The factor which stimulated the proliferation and outgrowth of the trophoblast was found in F > XM300 and XM300 > F > XMlOO but not in XMlOO > F > PM30 (Table 1). The minimum concentration of F > XM300 which induced the outgrowth of the trophoblast was found to be 0.03%. From this titration F > XM300 was estimated to contain 3300 units/ml (Table 1).
TABLE BIOASSAY DIFFERENTIATION
% of each fraction in BM
OF EMBRYO FACTOR
1 GROWTH 1 (EGDF-1)
AND IN FCS
% of embryos showing outgrowth” (per total embryos examined) after 2 days in culture F> XM300
XM300 > F>
XMlOO > F>PM30
XMlOO 10
4 2 1 0.5 0.25 0.12 0.06 0.03 0.15 0.008 units/ml a Classified
0 (7) 0 (6) 0 (7) 100 (6) 100 (8) 87 (7)
66 (6) 86 (7)
67 (9) 89 (9) 71 (7) 80 (5) 0 (8)
56 (9) 0 (6)
0 (10)
O(7) O(9)
0 (7) 3300
0
(6)
0 0 0 0 0 ---
(7) (7) (9) (4) (5)
800
as stage 8 and shown
FCS contains 330 units of EGDF-2. The ultrafiltrate permeable through PM 30 had no biological activity to promote egg cylinder growth. EGDF-3 Effects on the Development Embryo Proper and Yolk Sac
of the
The stage to which the embryo developed in various concentrations of HCS (Fig. 3). EGDF-2 Factor Present in the Lower Mo- Mouse blastocysts (stage 6) initially cullecular Weight Fraction of FCS tured in medium CMRL 1066 plus FCS for Mouse blastocysts at stage 6, if placed in 3 days reached stage 9. Then the medium FCS, developed to stage 8 after 2 days (Fig. was replaced by CMRL 1066 plus various 2A). At stage 8, if the culture medium was concentrations of HCS, from 1 to 40%. The culture medium was replaced daily by the replaced by BM, mouse embryos developed of HCS. Embryonly to stage 9 and no further (Fig. 2B). By particular concentration onic development was scored at the end of culturing stage 8 embryos in FCS or XMlOO > F > PM30 for 2 more days, they devel- 7 days’ incubation. In general, at low HCS concentrations, embryonic development oped to stage 10 (Fig. 2C). Thus, the factor was slower and less advanced than at higher which promotes the growth and differentiation of the egg cylinder is contained in the concentrations. After 4 days’ culture in 1% lower molecular fraction EGDF-2 and not HCS (Fig. 3A), embryonic development almost ceased at stage 9, with little increase in the higher molecular fraction EGDF-1 (Table 2). The minimum concentration of in embryonic mass. In 3% HCS for 4 days FCS which induced the growth of the egg (Fig. 3B), the embryos looked runted, alcylinder to stage 10 was found to be 0.3% though they reached stage 12. In 10% HCS (Table 3). Therefore, it is estimated that (Fig. 3C), the embryo proper differentiated
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FIG. 2. Embryo growth and differentiation factor (EGDF) 2. (A) Blastocysts (stage 6) were initially cultured in medium CMRL 1066 plus FCS. After 2 days in culture, blastocysts developed to stage 8. (B) At stage 8, the culture medium was replaced by BM and incubated further for 2 days. The inner cell mass increased and formed a small cavity in the center. The endodermaf cells covered the entire inner cell mass, which did not elongate (stage 9). (C) At stage 8, medium was replaced by BM plus XMlOO > F > PM30 or FCS and cultured further for 2 days. Mouse embryos increased in mass and elongated (stage 10).
poorly. In 40% HCS (Fig. 3D), the embryo proper developed very well, with a visible notochord, definite somites, a neural fold, and a pulsating heart. The biological activTABLE
2
BIOLOGICAL ACTIVITY OF EMBRYO GROWTH AND DIFFERENTIATION FACTOR 2 (EGDF-2) PRESENT IN THE LOWER MOLECULAR WEIGHT FRACTION OF FCS XM300 > XMlOO > FCS F> F > PM30” XMlOO No. of embryos* No. of embryos reaching stage 8 9 10 Stage lo/total embrvos
13
9
ity of EGDF-3 is heat sensitive and is not permeable through an Amicon Diaflo membrane XMlOO. Diameter of the yolk sac in various concentrations of HCS (Fig. 4). Blastocysts (stage 6) were initially cultured in CMRL 1066 plus FCS for 3 days. By the end of 3 days’ culture, the majority of embryos developed to stage 9. Then the medium was replaced thereafter by CMRL 1066 plus various concentrations of HCS, from 1 to
14 TABLE 3 BIOLOGICAL ASSAY FOR EMBRYO GROWTH AND DIFFERENTIATION FACTOR 2 (EGDF-2) IN FCS % of FCS in BM
9 4 0 O/13 = 0%
0 3 6
0 4 10
6/9 = 67% lo/14 = 71%
a The ultrafiltrate through PM30 has no activity for the induction of embryonic growth. b Total embryos contained in two culture plates.
10 5 2.5 1.2 0.6 0.3 0.15 0
Stages reached after 4 days of culture 8
9
1n
2 1 1 4 4 4 5 7
2 1 1 1 2 2 3 2
4 6 6 2 3 1 0 0
Stage IO/total embryos
4/8 6/8 6/8 2/17 3/9 l/7 O/8 o/9
= = = = = = = =
50% 75% 75% 29% 33% 14% 0% 0%
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Embryo Growth Factors of Mammals
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FIG. 3. Embryo growth and differentiation factor (EGDF) 3. Mouse blastocysts (stage 6) were cultured in medium CMRL 1066 plus FCS for 3 days. Then medium was replaced with BM plus (A) l%, (B) 3%, (C) IO%, and (D) 40% HCS. The culture medium was renewed daily with the same concentration of HCS. After 4 days in culture with HCS: (A) (1% HCS) A small embryonic cyst formed after stage 9 was retarded. (B) (3% HCS) A shrinking neural plate and a visible aflantois in a small visceral yolk sac cavity; runt embryo of stage 13. (C) (10% HCS) Larger visceral yolk sac cavity with degenerating neural plate and relatively well-developed allantois. (D) (40% HCS) A well-developed embryo proper which consisted of neural fold, notochord, and somites which were visible under a dissecting microscope (stage 15). Unstained live embryos. X 30.
40%. The medium was renewed daily with the same concentration of HCS. At the end of 8 days’ incubation from stage 6, the diameters of 15 embryos developed in each concentration of HCS were measured. The diameter of yolk sac developed during 8 days’ incubation was directly proportional to the concentration of HCS in the medium. The effects of different concentrations of HCS on the development of the embryo proper (Table 4). Mouse blastocysts (stage 6) were initially cultured in medium CMRL
1066 plus FCS for 3 days and 20% HCS for the next 2 days. By the fifth day of incubation, the majority of the embryos developed to stage 12. Then the medium was replaced by CMRL 1066 plus various specific concentrations of HCS, from 1.2 to 30%. This culture medium was replaced daily with the same concentration of HCS. Embryonic development was scored at the end of 8 days’ incubation. At high concentrations (20-30s) of HCS, the majority (6880%) of the embryos developed to stage 14
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TABLE 4 THE EFFECT OF THE HCS (EGDF-3) CONCENTRATION ON THE DEVELOPMENT OF EMBRYO PROPER Yolk sac or Small em% of stages 14 + younger than 15/t&d embryos/total HCS stage m/tobryos embryos in
BM 30 20 10 5 2.5 1.2 I of,
,
’ 3
’ 5
IO
20 CONCENTRATION
40 OF HCS
(%I
FIG. 4. The diameter of yolk sacs grown in various concentrations of HCS. The diameter of 15 embryos in each concentration of HCS was measured after 8 days of cultivation.
or 15, with a well-developed large embryo proper (as shown in Fig. 3D). Fifty-eight percent and 65% of the total number of embryos developed to a small undifferentiated embryo proper with a smaller yolk sac (Fig. 3C). In 1.2 to 2.5% HCS, the majority of the embryos (78100%) developed did not have an embryo proper or the embryos were less developed than stage 13 embryos (as shown in Fig. 3B). The proportion of cultures with complete normal-sized embryo proper was directly related to the concentration of HCS in the medium. At the lower concentration @i-10%) of HCS the majority of the embryos developed lacked an embryo proper or had only a small embryo proper, as shown in Fig. 3C. DISCUSSION
The preliminary results indicate that at least three factors successively regulate the continuous development of mouse embryos from stage 6 to stage 15 (Fig. 5). A series of tissue-specific macromolecules which promote the growth and differentiation of particular cell types of mammals has been identified, such as nerve growth factor (Bradshaw, 1978; Levi-Montalcini and Cal-
tal embryos
22/26 = 21/31= 3/20 = o/20 =
85% 68% 15% 0%
O/18 = 0% o/14 = 0%
O/26 = 5/31= 13/20 = 15/20= 4/18 = O/24 =
0% 16% 65% 58% 22% 0%
4/26 = 15% 5/31=16% 4/20 = 20% 11/26 = 42% 14/x3= 78% 14/14 = 100%
issano, 1979), epidermal growth factor (Carpenter and Cohen, 1979), macrophage-granulocyte inducer (Metcalf, 1977; Sachs, 1978), erythropoietin, fibroblast growth factor (Gospodarowicz et al., 1978), and angiogenic factor (Folkman and Cotran, 1976). Since embryonic development is a systematic continuous flow of stem cell differentiation, it is not surprising to find that certain steps of mammalian embryonic differentiation are regulated by exogenous macromolecules. The results also indicate that each stage of the continuous development of the mouse embryo is regulated successively by several specific molecules. Theoretically a lack or deficiency of any of the molecules during pregnancy could cause delayed development, embryonic runting, abortion, or malformation. Although resorption, some habitual abortions and congenital birth defects are attributed to genetic causes, the proximate cause of these maladies is not known. A continuous effort to demonstrate and identify any of the specific factors which promote the growth and differentiation of embryonic development is therefore important. It has been reported that some preparations of fetuin stimulated the outgrowth of mouse trophoblast (Gwatkin, 196613;Rizzino and Sherman, 1979). An attempt to identify growth factors for rat embryos has been initiated by New and Klein’s laboratory. Rat embryos explanted
beyond the egg-cylinder
stage developed in
YU-CHIH
Embryo Growth Factors
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of Mammals
473
days In culture
0
I
2
3
4
5
6
7
7.5
8-8.5
Witschl
6
7
8
9
IO
II
12
13
14
15
Theller
5
5
6
7
8
9
IO
II
I2
I3
3.5
4
4.5
5
5.5
6.5
7
7.5
8
8.5
stages gestation
CMRL
doys
:
1066 +
8SA
EGDF-I (FCS,
HCS)
FCS (EGDF-I+21
tics (EGDF-I+2+3)
(EGDF-I
(EGDF-I)
1
(EGDF-2)
(EGDF-2)
(EGDF-3)
FIG. 5. Stages of mouse embryos reached in various embryo growth and differentiation factors (EGDF). Blastocysts (stage 6 of Witschi, stage 5 of Theiler) in medium CMRL 1066 plus 0.25% BSA developed to stage 7 after 2 days of culture (Fig. 2A). Trophoblasts did not spread out on culture dishes, and embryos eventually died at stage 7. Blastocysts (stage 6 of Witschi) developed to stage 11 in medium CMRL 1066 plus 10% FCS after 5 days of culture. Fetal calf serum was separated into two fractions, a higher molecular weight fraction and a lower molecular weight fraction, by molecular sieving with Amicon’s DiafIo membranes XM300, XMlOO, and PM30. The larger molecular fraction is required for embryos to develop from stage 7 to stage 8 (EGDF-l), and the smaller molecular fraction promotes the embryonic growth from stage 8 to stage 11 (EGDF-2). Although some degree of embryonic growth continued beyond stage 11 in the medium using FCS as a sole source of macromolecules, the structure of the mouse embryos had become increasingly disproportional as compared to that developed in utero. Neural plates became atrophic and eventually disappeared. Mouse embryos resumed their normal growth to stage 15 in uitro, if FCS was replaced by HCS beyond stage 11. Therefore, HCS contains EGDF-3, which is indispensable for mouse embryos to grow from stage 11 to stage 15. Blastocysts (stage 6) were also able to develop to stage 15 in the medium containing HCS as a sole source of macromolecules. Therefore, HCS also contains EGDF-1 and EGDF-2.
rat serum for 2 to 4 days (New, 1978). When rat embryos were grown in serum obtained by “delayed centrifugation,” many of them acquired double hearts and other abnormalities, but in “immediately centrifuged” serum, they developed normally. Klein et al. have reported that cultures of lo-day rat embryos depleted a protein band with a molecular weight of approximately 125,000 from the rat serum medium. Delayed centrifuged serum differed from immediately centrifuged serum by a reduction in the 125,000 molecular weight protein
band due to the absence of two high molecular weight (>200,000) protein bands. Klein et al. (1978) have suggested that one of these proteins may be identical to the a2globulin of 125,000 molecular weight, identified in human serum as acidic DNA-binding proteins (Hoch and McVeg 1977). I wish to thank Dr. and Mrs. Frederik their help in editing the manuscript.
Bang for
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BRINSTER, R. (1967). Protein content of the mouse embryo during the fist five days of development. J. Reprod. Fertility 13.413-420. CARPENTER, G., and COHEN, S. (1979). Epidermal growth factor. Ann. Rev. Biochem. 48, 193-216. FOLKMAN, J., and COTRAN, R. (1976). Relation of vascular proliferation to tumor growth. Znt. Rev. Exp. Pathol; 16, 207-248. GONDA, M. A., and Hsu, Y-C. (1980). Correlative scanning electron, transmission electron, and light microscopic studies of the in vitro development of mouse embryos on a plastic substrate at the implantation stage. J. Embryol. Exp. Morphol. (in press). GOSPODAROWICZ, D., GREENBERG, G., BIALECKI, H., and ZETTER, B. R. (1978). Factors involved in the modulation of cell proliferation in vivo and in vitro: The role of fibroblast and epidermal growth factors in the proliferative response of mammalian cells. In Vitro 14,85-118. GWATKIN, R. B. L. (1966a). Amino acid requirements for attachment and outgrowth of the mouse blastocyst in vitro. J. Cell. Physiol. 68, 335-344. GWATKIN, R. B. L. (1966b). Defined media and development of mammalian eggs in vitro. Ann. N.Y. Acad. 5%. 139,79-90. HSU, Y. (1973). Differentiation in vitro of mouse embryos to the stage of early somite. Develop. Biol. 33, 403-411. Hsu, Y-C., BASKAR, J., STEVENS, L. C., and RASH, J. E. (1974). Development in vitro of mouse embryos from the two-cell egg stage to the early somite stage. J. Embryol. Exp. Morphol. 31, 235-245. Hsu, Y. (1979). In vitro development of individually cultured whole mouse embryos from blastocyst to early somite stage. Develop. Biol. 68, 453-461. JUURLINK, B. H., and FEDOROFF, S. (1977). Effects of culture milieu on the development of mouse blastocysts in vitro. In Vitro 13, 790-798. KLEIN, N. W., MINGHE’ITI, P. P., JACKSON, S. K., and VOGLER, M. A. (1978). Serum protein depletion by cultured rat embryos. J. Exp. Zool. 203, 313-318. LEVI-M• NTALCINI, R., and CALISSANO, P. (1979). The nerve growth factor. Sci. Amer. 240,68-77.
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LIBBUS, B., and Hsu, Y-C. (1980). Sequential development and tissue organization in whole mouse embryos cultured from blastocyst to early somite stage. Anat. Rec. (in press). MCLAREN, A., and HENSLEIGH, H. C. (1975). Culture of mammalian embryos over the implantation peof Mammals” riod. In “The Early Development (Balls and Wild, ed.), pp. 45-60. Cambridge University Press, London. METCALF, D. (1977). Hemopoietic Colonies, “Recent Results in Cancer Research,” Vol. 61, Springer-Verlag, Berlin, Heidelberg, New York. NEW, D. A. T. (1978). Whole-embryo culture and the study of mammalian embryos during organogenesis. Biol. Rev. 53, 81-122. PIENKOWSKI, M., SOLTER, D., and KOPROWSKI, H. (1974). Early mouse embryos: Growth and differentiation in vitro. Exp. Cell Res. 85,424-428. RIZZINO, A., and SHERMAN, M. I. (1979). Development and differentiation of mouse blastocysts in serumfree medium. Exp. Cell Res. 121,221-233. SACHS, L. (1978). Control of normal cell differentiation and the phenotypic reversion of malignancy in myeloid leukemia. Nature 274, 535-539. SHERMAN, M. I. (1975). The culture of cells derived from mouse blastocysta. Cell 5,343-349. SPINDLE, A., and PEDERSEN, R. A. (1973). Hatching, attachment and outgrowth of mouse blastocysts in vitro: Fixed nitrogen requirements. J. Exp. 2001. 186,305-318. THEILER, K. (1972). “The House Mouse.” SpringerVerlag, New York. WILEY, L. M., and PEDERSEN, R. A. (1977). Morphology of mouse egg cylinder development in vitro: A light and electron microscope study. J. Exp. 2001. 200,389-402. WILSON, I. B., and JENKINSON, E. I. (1974). Blastocyst differentiation in vitro. J. Reprod. Fert. 39, 243-
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BIOLOGY
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Embryo Growth and Differentiation Factors in Embryonic Mammals YU-CHIH
Sera of
Hsu
Laboratory of Mammalian Development and Oncogenesis, Department of Pathobiology, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland 21205 Received August 14, 1979; accepted in revised form December 5, 1979 Mouse embryos sequentially developed in vitro were classified by stages according to several established in vivo criteria. At least three heat-sensitive macromolecules from embryonic sera were found to be successively required for mouse embryos to continuously develop from stage 6 to stage 15 in vitro. Fetal calf serum (FCS) was found to be required for in vitro development only from stage 6 to stage 11, and human placental cord serum (HCS) was found essential from stage 11 to stage 15. The factors have been tentatively designated as embryo growth and differentiation factors (EGDF). FCS was separated by molecular sieving into at least two fractions: a large fraction (EGDF-1) and a smaller fraction (EGDF-2). EGDF-1 is required for embryos to develop from stage 7 to stage 8, EGDF-2 is required from stage 8 to stage 11. EGDF-3, the factor in human cord serum, was found to be indispensable for mouse embryonic development from stage 11 to stage 15. Since blastocysts (stage 6) were able to develop to stage 15 in the medium containing HCS as the sole source of macromolecules [Hsu, Y-C., et al. (1974). J. Embryol. Exp. Morphol. 31,235-2451, it is apparent that HCS also contains EGDF-1 and EGDF-2. Embryonic development was significantly retarded in decreasing concentrations of the three EGDFs. INTRODUCTION
Mouse embryos during the preimplantastage develop adequately in KrebsRinger’s bicarbonate solution with pyruvate, lactate, or glucose as a sole energy source for cleavage. These developing embryos during the cleavage stages do not require macromolecules or exogenous nitrogen for embryonic development. As a result, at the end of the cleavage stage, the total protein content of the whole embryos is decreased by 20% (Brinster, 1967). In contrast to the preimplantation stage, mouse embryos abruptly and exponentially increase their embryonic mass immediately after implantation. It has been reported that both amino acid and macromolecules are required beyond the implantation stage (Gwatkin, 1966a; Spindle and Pedersen, 1973). These drastic metabolic changes, from dependence on stored nutrients to an exogenous source uptake, are triggered by tion
the embryonic attachment at the implantation stage, thus offering many interesting problems in the field of implantation biology. It is intriguing to study the macromolecules which regulate the rapid growth and differentiation of embryonic development. On the other hand, it has been reported previously that it is now possible to develop mouse embryos in vitro from the two-cell egg stage (stage 2 of Witschi, 1972; Theiler, 1972) to the early somite stage (stage 15 of Wits&i and stage 13 of Theiler) (Hsu et al., 1974). Subsequently, after modification and improvement of the culture method, about 50-70% of the individually cultured blastocysts (stage 6) differentiated to stage 15 if there were frequent medium changes to remove embryonic waste (Hsu, 1979). The two-cell eggs of the mouse embryos were initially cultured in a chemically defined medium up to the blastocyst stage (stage 6). Then they were transferred to a commercial
culture
medium
which
contained
465 OOlZ-1606/80/060465-10$02.00/0 Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved
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calf serum or fetal calf serum. Blastocysts continued their growth after shedding the zona pellucida, attached to culture dishes, and formed egg cylinders. Several laboratories are now able to routinely culture mouse embryos from stage 6 to stage 11 with a commercial culture medium using fetal calf serum as a sole source of macromolecules (Pienkowski et al., 1974; Sherman, 1975; McLaren and Hensleigh, 1975; Juurlink and Federoff, 1977; Wiley and Pedersen, 1977). Because human placental cord serum is not commercially available, several laboratories had attempted to culture mouse embryos in vitro to the stage of early somite (stage 15) by using commercial fetal calf serum as a sole source of serum in the culture medium. Although some degree of embryonic growth continued beyond stage 11 in the medium using fetal calf serum as a sole source of macromolecules, the structure of the mouse embryos became increasingly distorted as compared to that developed in utero. The neural plate atrophied and eventually disappeared. These results were consistent. Mouse embryos, however, resumed their normal growth in vitro, if fetal calf serum was replaced by human placental cord serum beyond stage 11 (Hsu, 1973, 1979). These in vitro mouse embryos, at any stage between stages 6 and 15, were indistinguishable from those grown in utero to corresponding stages (Hsu et al., 1974;Wiley and Pedersen, 1977; Gonda and Hsu, 1980; Libbus and Hsu, 1980). These results suggested either that fetal calf serum lacks a factor for development of mouse embryos from stages 11 to 15 which is present in human placental cord serum or that fetal calf serum contains an inhibitor(s) against the factor in human placental cord serum or both. In order to attain the long-range goal of the study of the macromolecules which regulate rapid growth and differentiation of embryonic development, this paper reports
on some of the biological assay methods used to identify the macromolecules. MATERIALS
AND
METHODS
Sera Fetal calf serum (FCS’). The FCS (from Grand Island Biological Co., New York) is heat-inactivated at 56°C for 30 min and stored at -20°C until used. Human placental cord serum (HCS). Blood is withdrawn from the umbilical cord while the placenta is still in the uterus. The vacutainer containing cord blood is placed at 4’C. Cord blood is separated from blood clots by centrifugation at 4000 rpm for 30 min as soon as possible but not later than 24 hr after being withdrawn from the umbilical cord. Hemolyzed cord serum is discarded. Cord serum is heat-inactivated at 56°C for 30 min and used on that day or stored at -60°C and used within 2 days. Serum Fractionation by Amicon Diaflo Membrane Ultrafiltration Amicon Diaflo ultrafiltration membranes (XM300, XMlOO, XM50, PM30, and PMIO) were soaked in distilled water for at least 1 hr before use to remove coated glycerol. The membranes and support were then immersed in 5% formalin for sterilization with the glossy side toward the solution. After riming three times with 30 ml of sterile distilled water, membranes were placed on the supports and prefiltered with 10 ml of sterile distilled water two times to remove residual formalin. The stirred cell (Model 52 or 402) was sterilized by autoclaving without a supporting pad. Sera were ultrafiltered under 5% COZ and 95% Nz gas pressure of 10 to 20 psi. Fifty milliliters of heat’ Abbreviations used: FCS, fetal calf serum; HCS, human placental cord serum; BM, basal medium; BSA, bevin: serum albumin; EGDF, embryo growth and differentiation factor; F > XM300, FCS ultrafiltrate fraction not permeable through XM300, XM300 > F > XMlOO, FCS ultrafiltrate fraction permeable through XM300 but not through XMlW, XMlOO > F > PM30, FCS ultrafiltrate fraction permeable through XMlOO but not through PM30.
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inactivated fetal calf serum was ultrafiltered through the Amicon Diaflo membrane XM300 in the Model 52 stirred cell, and 45 ml of ultrafiltrate was obtained. Fifteen milliliters of medium CMRL 1066 was added to the 5 ml of retained fraction in the stirred cell (washout). The ultrafiltration was continued. This portion of ultrafiltrate was discarded. The “washout” with CMRL 1066 was repeated three times. The fetal calf serum fraction retained in membrane XM300 was designated F > XM300. The 45 ml of filtrate through XM300 was further ultrafiltered against XMlOO. The 35 ml of ultrafiltrate so obtained was designated fraction F < XMlOO. The fraction retained in the stirred cell was washed out three more times with 20 ml of medium CMRL 1066. The final portion retained in membrane XMlOO was designated XM300 > F > XMlOO. Preparation
of Blastocysts
Random bred CF 1 mice 6 weeks old were purchased from the Charles River Co., Massachusetts. Follicular growth was stimulated in the females by the injection of 5 IU of pregnant mare serum gonadotrophin (Organon, Inc., West Orange, New Jersey). Five IU of human chorionic gonadotropin was injected 48 hr later to stimulate ovulation. The female mice were then placed with males, one pair per cage. The next morning, the females were checked for the presence of a vaginal sperm plug (Day 0 of pregnancy). On the third day of pregnancy, they were killed by dislocation of the neck. All procedures in the preparation, cultivation, and observation of mouse embryos were conducted at an ambient temperature of 37°C under either a horizontal or a vertical flow hood. The uteri were removed from their mesometrium and placed in lOOmm plastic culture dishes. Using a dissecting microscope, each uterine horn was flushed with 1 ml of basal medium (BM) which consisted of medium CMRL 1066 plus 0.25% bovine serum albumin (BSA)
467
(Miles Laboratories, Inc.) Blastocysts were sucked into a capillary pipet and pooled in a 35-mm plastic culture dish containing 2 ml of basal medium; the dish was constantly flushed with 5% COZ and 95% air to keep the pH of the medium at 7.4. Ten blastocysts were distributed by capillary pipet into separate culture dishes, each of which contained 2 ml of BM. The cultures were maintained at 37°C in humidified incubators containing 5% COZ and 95% air. The concentration of CO2 was automatically regulated. Biological Assay of Embryo Growth Differentiation Factors (EGDF)
and
Since the biological activities which promote the growth and differentiation of mouse embryos have not been identified, the term “factor” will be tentatively used for convenience in this paper. Biological assay of EGDF-1. Mouse blastocysts were flushed out of the uterine horns with 1 ml of BM. A fraction of FCS was serially diluted with 2 ml of BM in 35mm Flacon tissue cultures dishes. Ten blastocysts were placed in each culture dish. After 2 days of incubation, when most of the blastocysts had shed the zona pellucida, the blastocysts were scored into three categories: (1) blastocysts floating on culture medium and not attached; (2) blastocysts slightly attached to culture dishes but trophoblasts not spread out (Fig. 1A) (both (1) and (2) are classified as stage 7); and (3) blastocysts attached and trophoblasts spread out on culture dishes (stage 8) (Fig. 1D). Biological assay of EGDF-2. About 10 blastocysts (stage 6) were initially cultured in 35-mm culture dishes which contained 2 ml of medium CMRL 1066 plus 10% fetal calf serum. After 2 days, when the blastocysts had attached to the culture dishes, the culture medium was removed and rinsed once with 2 ml of BM. Then the attached blastocysts at stage 8 were further cultured in BM plus a serial dilution of each
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FIG. 1. Embryo growth and differentiation factor (EGDF) 1. (A) Blastocysts (stage 6) were cultured in BM, which consists of medium CMRL 1066 plus 0.25% BSA. After 2 days in culture, blastocysts shed the zona pellucida and attached lightly to the surface of the culture dishes. Mural trophoblasts, however, did not spread out (stage 7). (B) After 3 days in culture. (C) After 4 days in BM, trophoblasts began to degenerate. (D) Fetal calf serum or F > XM300 was added in BM before trophoblasts degenerated, or blastocysts (stage 6) were cultured in BM for 2 days; trophoblasts spread out on culture dishes, leaving an inner cell mass on the center of the trophoblast cell sheet (stage 8). X 400.
fraction of serum ultrafiltrate or FCS. After further incubation for 2 days, the embryos were scored and classified by developmental stages as stages 8,9, 10, and 11. Biological assay of EGDF-3. Blastocysts were initially cultured in 2 ml of medium CMRL 1066 plus 10% FCS for 3 days. Then the medium was replaced by 2 ml of BM which contained different serial concentrations of HCS in different cultures. The medium in each case was renewed daily with the same concentration of HCS. Embryos
were again scored and classified at the end of 7 days’ cultivation. RESULTS
The mouse embryos which developed in vitro were classified into stages according to the in uiuo criteria of Witschi (1972), Theiler
(1972), and Hsu (1979).
EGDF-1 Mouse blastocysts were initially cultured in BM consisting of medium CMRL 1066
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plus 0.25% BSA. When blastocysts (stage 6) were cultured in BM, they shed their zona pellucida and attached lightly to the culture dishes (stage 7) after 2 days of culture (Fig. 1A). The trophoectoderm, however, did not proliferate or spread out on the surface of the culture dishes. Blastocysts remained in stage 7 (Fig. 1B) for 2 or 3 days if kept in BM, and then gradually disintegrated (Fig. 1C). If BSA was replaced by FCS before the blastocysts had died, the trophoblast resumed proliferation and spread out (Fig. 1D). Fetal calf serum was fractionated into three fractions-two higher molecular weight fractions, F > XM300 and XM300 > F > XMlOO, and a lower molecular fraction, XMlOO > F > PM30-by ultrafiltration through Amicon Diaflo membranes XM300, XMlOO, and PM30. The factor which stimulated the proliferation and outgrowth of the trophoblast was found in F > XM300 and XM300 > F > XMlOO but not in XMlOO > F > PM30 (Table 1). The minimum concentration of F > XM300 which induced the outgrowth of the trophoblast was found to be 0.03%. From this titration F > XM300 was estimated to contain 3300 units/ml (Table 1).
TABLE BIOASSAY DIFFERENTIATION
% of each fraction in BM
OF EMBRYO FACTOR
1 GROWTH 1 (EGDF-1)
AND IN FCS
% of embryos showing outgrowth” (per total embryos examined) after 2 days in culture F> XM300
XM300 > F>
XMlOO > F>PM30
XMlOO 10
4 2 1 0.5 0.25 0.12 0.06 0.03 0.15 0.008 units/ml a Classified
0 (7) 0 (6) 0 (7) 100 (6) 100 (8) 87 (7)
66 (6) 86 (7)
67 (9) 89 (9) 71 (7) 80 (5) 0 (8)
56 (9) 0 (6)
0 (10)
O(7) O(9)
0 (7) 3300
0
(6)
0 0 0 0 0 ---
(7) (7) (9) (4) (5)
800
as stage 8 and shown
FCS contains 330 units of EGDF-2. The ultrafiltrate permeable through PM 30 had no biological activity to promote egg cylinder growth. EGDF-3 Effects on the Development Embryo Proper and Yolk Sac
of the
The stage to which the embryo developed in various concentrations of HCS (Fig. 3). EGDF-2 Factor Present in the Lower Mo- Mouse blastocysts (stage 6) initially cullecular Weight Fraction of FCS tured in medium CMRL 1066 plus FCS for Mouse blastocysts at stage 6, if placed in 3 days reached stage 9. Then the medium FCS, developed to stage 8 after 2 days (Fig. was replaced by CMRL 1066 plus various 2A). At stage 8, if the culture medium was concentrations of HCS, from 1 to 40%. The culture medium was replaced daily by the replaced by BM, mouse embryos developed of HCS. Embryonly to stage 9 and no further (Fig. 2B). By particular concentration onic development was scored at the end of culturing stage 8 embryos in FCS or XMlOO > F > PM30 for 2 more days, they devel- 7 days’ incubation. In general, at low HCS concentrations, embryonic development oped to stage 10 (Fig. 2C). Thus, the factor was slower and less advanced than at higher which promotes the growth and differentiation of the egg cylinder is contained in the concentrations. After 4 days’ culture in 1% lower molecular fraction EGDF-2 and not HCS (Fig. 3A), embryonic development almost ceased at stage 9, with little increase in the higher molecular fraction EGDF-1 (Table 2). The minimum concentration of in embryonic mass. In 3% HCS for 4 days FCS which induced the growth of the egg (Fig. 3B), the embryos looked runted, alcylinder to stage 10 was found to be 0.3% though they reached stage 12. In 10% HCS (Table 3). Therefore, it is estimated that (Fig. 3C), the embryo proper differentiated
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FIG. 2. Embryo growth and differentiation factor (EGDF) 2. (A) Blastocysts (stage 6) were initially cultured in medium CMRL 1066 plus FCS. After 2 days in culture, blastocysts developed to stage 8. (B) At stage 8, the culture medium was replaced by BM and incubated further for 2 days. The inner cell mass increased and formed a small cavity in the center. The endodermaf cells covered the entire inner cell mass, which did not elongate (stage 9). (C) At stage 8, medium was replaced by BM plus XMlOO > F > PM30 or FCS and cultured further for 2 days. Mouse embryos increased in mass and elongated (stage 10).
poorly. In 40% HCS (Fig. 3D), the embryo proper developed very well, with a visible notochord, definite somites, a neural fold, and a pulsating heart. The biological activTABLE
2
BIOLOGICAL ACTIVITY OF EMBRYO GROWTH AND DIFFERENTIATION FACTOR 2 (EGDF-2) PRESENT IN THE LOWER MOLECULAR WEIGHT FRACTION OF FCS XM300 > XMlOO > FCS F> F > PM30” XMlOO No. of embryos* No. of embryos reaching stage 8 9 10 Stage lo/total embrvos
13
9
ity of EGDF-3 is heat sensitive and is not permeable through an Amicon Diaflo membrane XMlOO. Diameter of the yolk sac in various concentrations of HCS (Fig. 4). Blastocysts (stage 6) were initially cultured in CMRL 1066 plus FCS for 3 days. By the end of 3 days’ culture, the majority of embryos developed to stage 9. Then the medium was replaced thereafter by CMRL 1066 plus various concentrations of HCS, from 1 to
14 TABLE 3 BIOLOGICAL ASSAY FOR EMBRYO GROWTH AND DIFFERENTIATION FACTOR 2 (EGDF-2) IN FCS % of FCS in BM
9 4 0 O/13 = 0%
0 3 6
0 4 10
6/9 = 67% lo/14 = 71%
a The ultrafiltrate through PM30 has no activity for the induction of embryonic growth. b Total embryos contained in two culture plates.
10 5 2.5 1.2 0.6 0.3 0.15 0
Stages reached after 4 days of culture 8
9
1n
2 1 1 4 4 4 5 7
2 1 1 1 2 2 3 2
4 6 6 2 3 1 0 0
Stage IO/total embryos
4/8 6/8 6/8 2/17 3/9 l/7 O/8 o/9
= = = = = = = =
50% 75% 75% 29% 33% 14% 0% 0%
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FIG. 3. Embryo growth and differentiation factor (EGDF) 3. Mouse blastocysts (stage 6) were cultured in medium CMRL 1066 plus FCS for 3 days. Then medium was replaced with BM plus (A) l%, (B) 3%, (C) IO%, and (D) 40% HCS. The culture medium was renewed daily with the same concentration of HCS. After 4 days in culture with HCS: (A) (1% HCS) A small embryonic cyst formed after stage 9 was retarded. (B) (3% HCS) A shrinking neural plate and a visible aflantois in a small visceral yolk sac cavity; runt embryo of stage 13. (C) (10% HCS) Larger visceral yolk sac cavity with degenerating neural plate and relatively well-developed allantois. (D) (40% HCS) A well-developed embryo proper which consisted of neural fold, notochord, and somites which were visible under a dissecting microscope (stage 15). Unstained live embryos. X 30.
40%. The medium was renewed daily with the same concentration of HCS. At the end of 8 days’ incubation from stage 6, the diameters of 15 embryos developed in each concentration of HCS were measured. The diameter of yolk sac developed during 8 days’ incubation was directly proportional to the concentration of HCS in the medium. The effects of different concentrations of HCS on the development of the embryo proper (Table 4). Mouse blastocysts (stage 6) were initially cultured in medium CMRL
1066 plus FCS for 3 days and 20% HCS for the next 2 days. By the fifth day of incubation, the majority of the embryos developed to stage 12. Then the medium was replaced by CMRL 1066 plus various specific concentrations of HCS, from 1.2 to 30%. This culture medium was replaced daily with the same concentration of HCS. Embryonic development was scored at the end of 8 days’ incubation. At high concentrations (20-30s) of HCS, the majority (6880%) of the embryos developed to stage 14
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TABLE 4 THE EFFECT OF THE HCS (EGDF-3) CONCENTRATION ON THE DEVELOPMENT OF EMBRYO PROPER Yolk sac or Small em% of stages 14 + younger than 15/t&d embryos/total HCS stage m/tobryos embryos in
BM 30 20 10 5 2.5 1.2 I of,
,
’ 3
’ 5
IO
20 CONCENTRATION
40 OF HCS
(%I
FIG. 4. The diameter of yolk sacs grown in various concentrations of HCS. The diameter of 15 embryos in each concentration of HCS was measured after 8 days of cultivation.
or 15, with a well-developed large embryo proper (as shown in Fig. 3D). Fifty-eight percent and 65% of the total number of embryos developed to a small undifferentiated embryo proper with a smaller yolk sac (Fig. 3C). In 1.2 to 2.5% HCS, the majority of the embryos (78100%) developed did not have an embryo proper or the embryos were less developed than stage 13 embryos (as shown in Fig. 3B). The proportion of cultures with complete normal-sized embryo proper was directly related to the concentration of HCS in the medium. At the lower concentration @i-10%) of HCS the majority of the embryos developed lacked an embryo proper or had only a small embryo proper, as shown in Fig. 3C. DISCUSSION
The preliminary results indicate that at least three factors successively regulate the continuous development of mouse embryos from stage 6 to stage 15 (Fig. 5). A series of tissue-specific macromolecules which promote the growth and differentiation of particular cell types of mammals has been identified, such as nerve growth factor (Bradshaw, 1978; Levi-Montalcini and Cal-
tal embryos
22/26 = 21/31= 3/20 = o/20 =
85% 68% 15% 0%
O/18 = 0% o/14 = 0%
O/26 = 5/31= 13/20 = 15/20= 4/18 = O/24 =
0% 16% 65% 58% 22% 0%
4/26 = 15% 5/31=16% 4/20 = 20% 11/26 = 42% 14/x3= 78% 14/14 = 100%
issano, 1979), epidermal growth factor (Carpenter and Cohen, 1979), macrophage-granulocyte inducer (Metcalf, 1977; Sachs, 1978), erythropoietin, fibroblast growth factor (Gospodarowicz et al., 1978), and angiogenic factor (Folkman and Cotran, 1976). Since embryonic development is a systematic continuous flow of stem cell differentiation, it is not surprising to find that certain steps of mammalian embryonic differentiation are regulated by exogenous macromolecules. The results also indicate that each stage of the continuous development of the mouse embryo is regulated successively by several specific molecules. Theoretically a lack or deficiency of any of the molecules during pregnancy could cause delayed development, embryonic runting, abortion, or malformation. Although resorption, some habitual abortions and congenital birth defects are attributed to genetic causes, the proximate cause of these maladies is not known. A continuous effort to demonstrate and identify any of the specific factors which promote the growth and differentiation of embryonic development is therefore important. It has been reported that some preparations of fetuin stimulated the outgrowth of mouse trophoblast (Gwatkin, 196613;Rizzino and Sherman, 1979). An attempt to identify growth factors for rat embryos has been initiated by New and Klein’s laboratory. Rat embryos explanted
beyond the egg-cylinder
stage developed in
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473
days In culture
0
I
2
3
4
5
6
7
7.5
8-8.5
Witschl
6
7
8
9
IO
II
12
13
14
15
Theller
5
5
6
7
8
9
IO
II
I2
I3
3.5
4
4.5
5
5.5
6.5
7
7.5
8
8.5
stages gestation
CMRL
doys
:
1066 +
8SA
EGDF-I (FCS,
HCS)
FCS (EGDF-I+21
tics (EGDF-I+2+3)
(EGDF-I
(EGDF-I)
1
(EGDF-2)
(EGDF-2)
(EGDF-3)
FIG. 5. Stages of mouse embryos reached in various embryo growth and differentiation factors (EGDF). Blastocysts (stage 6 of Witschi, stage 5 of Theiler) in medium CMRL 1066 plus 0.25% BSA developed to stage 7 after 2 days of culture (Fig. 2A). Trophoblasts did not spread out on culture dishes, and embryos eventually died at stage 7. Blastocysts (stage 6 of Witschi) developed to stage 11 in medium CMRL 1066 plus 10% FCS after 5 days of culture. Fetal calf serum was separated into two fractions, a higher molecular weight fraction and a lower molecular weight fraction, by molecular sieving with Amicon’s DiafIo membranes XM300, XMlOO, and PM30. The larger molecular fraction is required for embryos to develop from stage 7 to stage 8 (EGDF-l), and the smaller molecular fraction promotes the embryonic growth from stage 8 to stage 11 (EGDF-2). Although some degree of embryonic growth continued beyond stage 11 in the medium using FCS as a sole source of macromolecules, the structure of the mouse embryos had become increasingly disproportional as compared to that developed in utero. Neural plates became atrophic and eventually disappeared. Mouse embryos resumed their normal growth to stage 15 in uitro, if FCS was replaced by HCS beyond stage 11. Therefore, HCS contains EGDF-3, which is indispensable for mouse embryos to grow from stage 11 to stage 15. Blastocysts (stage 6) were also able to develop to stage 15 in the medium containing HCS as a sole source of macromolecules. Therefore, HCS also contains EGDF-1 and EGDF-2.
rat serum for 2 to 4 days (New, 1978). When rat embryos were grown in serum obtained by “delayed centrifugation,” many of them acquired double hearts and other abnormalities, but in “immediately centrifuged” serum, they developed normally. Klein et al. have reported that cultures of lo-day rat embryos depleted a protein band with a molecular weight of approximately 125,000 from the rat serum medium. Delayed centrifuged serum differed from immediately centrifuged serum by a reduction in the 125,000 molecular weight protein
band due to the absence of two high molecular weight (>200,000) protein bands. Klein et al. (1978) have suggested that one of these proteins may be identical to the a2globulin of 125,000 molecular weight, identified in human serum as acidic DNA-binding proteins (Hoch and McVeg 1977). I wish to thank Dr. and Mrs. Frederik their help in editing the manuscript.
Bang for
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LIBBUS, B., and Hsu, Y-C. (1980). Sequential development and tissue organization in whole mouse embryos cultured from blastocyst to early somite stage. Anat. Rec. (in press). MCLAREN, A., and HENSLEIGH, H. C. (1975). Culture of mammalian embryos over the implantation peof Mammals” riod. In “The Early Development (Balls and Wild, ed.), pp. 45-60. Cambridge University Press, London. METCALF, D. (1977). Hemopoietic Colonies, “Recent Results in Cancer Research,” Vol. 61, Springer-Verlag, Berlin, Heidelberg, New York. NEW, D. A. T. (1978). Whole-embryo culture and the study of mammalian embryos during organogenesis. Biol. Rev. 53, 81-122. PIENKOWSKI, M., SOLTER, D., and KOPROWSKI, H. (1974). Early mouse embryos: Growth and differentiation in vitro. Exp. Cell Res. 85,424-428. RIZZINO, A., and SHERMAN, M. I. (1979). Development and differentiation of mouse blastocysts in serumfree medium. Exp. Cell Res. 121,221-233. SACHS, L. (1978). Control of normal cell differentiation and the phenotypic reversion of malignancy in myeloid leukemia. Nature 274, 535-539. SHERMAN, M. I. (1975). The culture of cells derived from mouse blastocysta. Cell 5,343-349. SPINDLE, A., and PEDERSEN, R. A. (1973). Hatching, attachment and outgrowth of mouse blastocysts in vitro: Fixed nitrogen requirements. J. Exp. 2001. 186,305-318. THEILER, K. (1972). “The House Mouse.” SpringerVerlag, New York. WILEY, L. M., and PEDERSEN, R. A. (1977). Morphology of mouse egg cylinder development in vitro: A light and electron microscope study. J. Exp. 2001. 200,389-402. WILSON, I. B., and JENKINSON, E. I. (1974). Blastocyst differentiation in vitro. J. Reprod. Fert. 39, 243-
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E. (1972). Characterization of developmental Part II. “Rat, Biology Data Book,” 2nd ed., pp. 178-180. Federation of American SocieExperimental Biology.