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Na+-dependent amino acid transport in preimplantation mouse embryos
DEVELOPMENTALBIOLOGY46, 1%-‘c?o1(19'%)
Na+-Dependent
Amino
Acid Transport
Mouse II. Metabolic
Inhibitors
in Preimplantation
Embryos
and Nature
of the Cation
Requirement
RAYMOND M. BORLAND’ AND RICHARD J. TASCA~ Department
of Biological
Sciences, University Accepted
of Delaware,
Newark,
Delaware
19711
May 7, 1975
Experiments were conducted in order to determine the energy source and nature of the cation dependency of [aH]methionine transport in preimplantation mouse embryos. The energy source of methionine transport was studied at the late four-cell and early blastocyst stages. The embryos, raised in vitro, were incubated for 1 hr in inhibitor(s) of energy metabolism and then transferred for 1 hr to medium that contained inhibitor(s) and *H-methionine. These inhibitor studies suggest that respiration and glycolysis are needed to maintain uptake of methionine in early blastocysts. Late four-cell embryos seem to utilize respiration alone for transport. The cation dependency of methionine transport was studied at the late morula and early blastocyst stages. The kinetics of methionine uptake by early blastocysts in Na+-depleted media indicate a competitive type of inhibition. The uptake of methionine by early blastocysts is relatively resistant to ouabain and unaffected by K+-free medium. In contrast, methionine uptake by late morula-stage embryos is markedly inhibited by ouabain and K+-free medium in 1 hr. These results suggest that 1) Na+ serves to increase the affinity of methionine for the carrier in early blastocysts, 2) the cation gradients do not supply a major fraction of the energy required for methionine transport, and/or the gradients are difficult to perturb once the blastocyst has formed, and 3) putative Na+ pumps may be localized on the blastocoelic surface of the blastocysts. INTRODUCTION
Recent studies have shown that the permeability of preimplantation stage mouse embryos to exogenously supplied amino acids increases at the eight-cell, morula and blastocyst stages (Tasca and Hillman, 1970; Brinster, 1971; Epstein and Smith, 1973). It is also known that late four-cell embryos and early blastocysts can concentrate leucine (Epstein and Smith, 1973) and methionine (Borland and Tasca, 1974) and transport methionine and leutine by processes that exhibit saturable, Michaelis-Menten-type kinetics (Borland and Tasca, 1974). Additional experiments demonstrated amino acid competition for ‘Present address: Department of Physiology and Laboratory of Human Reproduction and Reproductive Biology, Harvard Medical School, 45 Shattuck Street, Boston, MA 02115. ‘To whom requests for reprints should he addressed.
transport and temperature dependency of amino acid transport which are also characteristic of carrier-mediated, active transport systems (Epstein and Smith, 1973; Borland and Tasca, 1974). The transport of leucine and methionine at the late four-cell stage is Na+ independent, whereas transport of these two amino acids is completely Na+ dependent at the early blastocyst stage (Borland and Tasca, 1974). In other experimental systems, Na+ and K+ gradients have been proposed as sources of energy for amino acid transport (Vidaver, 1964a, b, c; review, Schultz and Curran, 1970). Other studies argue that cellular energy metabolism directly provides the major source of energy for amino acid transport (Tucker and Kimmich, 1973; Kimmich and Randles, 1973a, b; Lin and Johnstone, 1971). The present studies were undertaken in order to determine the source of energy for 192
Copyright 0 1975 by AcademicPress,Inc. AU rights of reproductionin any form reserved.
BORLAND
AND TASCA
Methionine
amino acid transport in preimplantation mouse embryos and to further characterize the Na+ dependency of transport. Therefore, experiments were designed to determine the effects upon amino acid transport at the late four-cell, morula and blastocyst stages of 1) inhibitors of energy metabolism and 2) Na+ concentration, K+-free media, and ouabain. MATERIALS
Superovulation
AND
METHODS
and
embryo
culture.
Eight- to twelve-week-old random-bred Swiss female mice (Flow Laboratory and Charles Rver Breeding Laboratories) were superovulated with 10 IU of pregnant mare serum gonadotropin (Gestyl, Organon, Inc.) given ip, followed 44 hr later by 10 IU of human chorionic gonadotropin, ip (Pregnyl, Organon, Inc.) (Edwards and Gates, 1959). The females were mated following the second injection, and those with vaginal plugs were selected as pregnant 21-23 hr later. The pregnant females were sacrificed by cervical dislocation, and the twocell embryos collected from the oviducts on the day following vaginal plug observation. The embryos were raised in vitro according to the method of Brinster (1963) in Brinster’s medium for ovum culture (BMOC) (Brinster, 1965) under light-weight paraffin oil (Fisher 125/135). Late four-cell embryos were used for experiments after 14-20 hr in vitro. Morulae and early blastocysts had been in vitro for 46-52 hr by the time of experimentation. Blastocysts at identical stages of development, as measured by the degree of blastocyst expansion, were used for each series of experiments. Early blastocysts contained 20-30 cells as determined by acetic-orcein staining. Labeling procedures. The completely defined medium (FMOCG, Ficoll medium for ovum culture with glucose) used during preincubation and labeling periods consisted of Brinster’s medium for ovum culture (BMOC, Brinster, 1965) but without bovine serum albumin, which was replaced
Uptake in Mouse Embryos
193
by 1 g/liter of Ficoll (Pharmacia), a nonmetabolizable, synthetic, sucrose polymer, and containing 0.1% glucose (Fisher Scientific Co.). In the preincubation inhibitor studies (Table l), late four-cell and early blastocyst-stage embryos were incubated for 1 hr in inhibitor(s) and then transferred for 1 hr to medium which contained inhibitor(s) and [sH]methionine. The experimental culture medium for labeling was prepared by adding [sH]methionine to FMOCG. Commercially prepared L- [methyl-sH]methionine, sp act, 4.02 Ci/mmole, sp act, and L- [3-3H(N)]phenylalanine, 5.39, were obtained from New England Nuclear Corporation. The inhibitors used in this series of studies were purchased from the following companies: Iodoacetic acid (Eastman Organic Chemicals); 2,4dinitrophenol (DNP) (Fisher Scientific Co.); potassium cyanide (KCN) (J. T. Baker Chemical Co., Phillipsburg, NJ); oligomycin (15% oligomycin A and 85% oligomycin B, Sigma Chemical Co.); and strophanthin G (ouabain, Sigma Chemical Co.). Twenty early blastocysts, 20 late morulae, or 30 late four-cell embryos were incubated per experiment. One drop of medium (Vi-Y2 ml) was placed in a 60 x 15-mm tissue culture dish (Falcon Plastics R3002). Embryos to be preincubated and labeled were selected randomly and transferred in lo-20 ~1 of BMOC in a micropipet directly to the drop of experimental medium. Light-weight paraffin oil was not used to cover the drops of experimental media due to the lipid solubility of some of the inhibitors. The embryos were incubated during the experimental treatment in high humidity in 5% CO, and 95% air at 37°C unless stated otherwise. After incubation the embryos were transferred sequentially in a micropipet, as stated above, through four changes of ice-cold (0-4°C) BMOC under light-weight paraffin oil, and immediately frozen in a tissue culture tube in an acetone-solid CO, bath.
194
DEVELOPMENTAL
BIOL~C:Y
The Na+-depleted medium (25 mM Na+) was prepared by modifying BMOC (Brinster, 1965) as follows: Choline chloride (Sigma Chemical Co.) equimolarly replaced NaCl, K-lactate replaced Na-lactate, K-pyruvate replaced Na-pyruvate, 1 mg/ml of Ficoll replaced bovine serum albumin, and the medium contained 0.1% glucose. K-lactate was prepared as follows: 1.82 ml of concentrated lactic acid (m-lactic acid, free acid, Sigma Chemical Co.) was added to 200 ml of double-distilled water, neutralized to pH 7.4 (about 15 ml of 1 N KOH), and added to a final volume of 1 liter of medium. Various concentrations of Na+-containing medium were then obtained by appropriately diluting Na+depleted medium with FMOCG. Thus, normal BMOC contains 5.97 mM K+, the Na+-depleted medium contains 26.97 mM K+. K+ concentrations as high as this do not affect normal embryonic development (Wales, 1970) and are normally present in the mouse oviduct (Roblero, Biggers and Lechene, personal communication). Extraction procedures. The extraction procedures were a modification of the procedures of Brinster (1971). The sample tubes were stored at -20°C until extraction. One milligram of bovine serum albumin and 1 mg of cold carrier amino acid in 20 ~1 of water were added to each sample. The tubes were then freezethawed four times with mild agitation during each thawing to insure complete breakdown of cell structures. One hundred microliters of ice-cold 10% trichloroacetic acid (TCA) was added to each tube, the contents gently mixed, the tubes stored on ice for 10 min, and then centrifuged at 10,000 rpm for 10 min in a Sorvall RC-2 centrifuge (SS-34 rotor). The TCA supernatant fluid was drawn off with a finely drawn Pasteur pipet and saved in a scintillation vial (TCA-soluble fraction). The TCA precipitate was then resuspended in 200 ~1 of 5% TCA, the tube vortexed gently, and then stored on ice for 10 min. The
Vo~m1~46,1975
tubes were again centrifuged at 10,000 rpm in a Sorvall RC-2 and the supernatant fluid removed and pooled with the soluble fraction. The precipitate was dissolved in 100 ~1 of 10% KOH for 30 min and was then transferred to a scintillation vial (TCAinsoluble fraction). Ten milliliters of Cocktail D (100 g of naphthalene, 5 g of 2,5diphenyloxazole in 1 liter of p-dioxane) were added to each vial, and the vials were counted on a Beckman liquid scintillation counter (LS-230). Counting efficiencies (35-40%) were determined by the method of internal standards, and all values are expressed in dpm/embryo/time interval. FtESULTS
Effects
of Inhibitors
on the
Uptake
of
Various inhibitors (oligomycin, 5 x 10ms g/ml; 2,4-dinitrophenol, 1O-s-1O-4 M, and KCN, lo-* M) were used in initial experiments to determine the source(s) of energy for methionine transport in early blastocyst stage mouse embryos. Embryos were incubated for 1 hr simultaneously in FMOCG with [sH]methionine and one of the above inhibitors. Under these conditions, methionine transport was not inhibited. It is possible that the 1 hr incubation does not sufficiently deplete the endogenous energy supply to affect methionine transport. Therefore, a series of preincubation experiments (Table 1) were conducted in which early blastocysts were incubated for 1 hr in inhibitor(s) and then transferred for 1 hr to medium which contains inhibitor(s) and [sH]methionine. The uptake of [sH]methionine (1.25 x 1OT8 M) has been shown to be linear for 1 hr at both the late four-cell and early blastocyst stages (Borland and Tasca, 1974). Culture medium droplets were not placed under oil for these experiments because of the high partition coefficient of some of the inhibitors. In view of the known changes during preimplantation mouse development in energy metabolism (Biggers, 1971), glycogen lev-
BORLAND
AND TAWA
Methionine TABLE
EFFECT OF INHIBITORY
ON [‘HIMETHIONINE
UPTAKE
1 IN FOUR-CELL EMBRYOS AND EARLY BLASTOCYSTS”
Early blastocyst
Late four cell
Treatment
TCA-soluble fraction
Percent of control
(dpm/embryo/hr) Control Ouabain (lo-’ IW) KCN (10-l iI!) Iodoacetic acid (lo-‘Ikf) 2,4-Dinitrophenol (lO-’ M Oligomycin (5 x lo-’ g/ml) Iodoacetic acid (lOma I@) + oligomycin (5 x 10--Bg/ml)
195
Uptake in Mouse Embryos
119.5 * 4.9 138.3 + 17.7 163.5 + 16.6 183.3 zt 16.6” 53.8 + 8.2’ 48.4 + 13.2b
TCA-soluble fraction
Percent of control
(dpm/embryo/hr) 100 116 137 153 45 41
537.1 385.7 566.7 107.0 576.4 240.7 38.8
zt 43.8 + 20.3b zt 8.4 zt 12.7c z+z27.8 f 35.2’ + 6.6’
100 72 106 20 107 45 7
“Thirty late four-cell or 20 early blastocyst-stage mouse embryos were preincubated in FMOCG with inhibitor(s) for 1 hr, and then incubated for an additional 1 hr in FMOCG with inhibitor(s) containing 1.25 x 10ee A4 L- [methyl-3H]methionine. After labeling, the embryos were washed, frozen, TCA-extracted, and the TCA-soluble and -insoluble fractions counted. The values are the mean + the standard error of the mean. The culture media drops were not placed under light paraffin oil in these experiments owing to the high lipid solubilitv of some of the inhibitors. The significance of the differences between samples was determined using Student’s t-test. b Significant at P < 0.01. c Significant at P < 0.001.
els (Ozias and Stern, 1973), ATP content and metabolism (Quinn and Wales, 1971, 1973; Ginsberg and Hillman, 1973), mitochondrial structure (Hillman and Tasca, 1969; Stern et al. 1971) and 0, consumption (Mills and Brinster, 1967), identical experiments were conducted on late fourcell embryos to compare the energetics of methionine transport between the two stages. As shown in Table 1, the uptake of methionine in late four-cell embryos is not significantly affected by ouabain (10m3M), an inhibitor of Na+-K+ ATPase. Iodoacetate (10e3 M), an inhibitor of glyceraldehyde-P-dehydrogenase, and 2,4-dinitrophenol (DNP) (lo-’ M), an uncoupler of oxidative phosphorylation, do not inhibit uptake. In fact, DNP (10-l M) significantly stimulates uptake, while inhibiting incorporation by 60% (unpublished). Oligomycin (5 x lOmeg/ml), an inhibitor of respiration (Lardy et al., 1964), decreases uptake to 45% of the control value. This inhibition was not further decreased by the simultaneous presence of iodoacetate and
oligomycin, suggesting that glycolysis is not an important source of energy for methionine transport at the four-cell stage. In the early blastocyst-stage mouse embryo, ouabain ( 1O-3M) decreases uptake of [3H]methionine to approximately 72% of the control value (Table 1). Iodoacetate (lOma M) markedly decreases uptake (to 20% of control value). KCN (10e2 M), an inhibitor of cytochrome oxidase, and DNP (lo-’ M) do not significantly affect uptake of methionine in early blastocysts. Oligomycin (5 x 1Om8g/ml) markedly inhibits both uptake and incorporation (unpublished) but to different degrees (55 and 95%, respectively). This virtual cessation of incorporation indicates that oligomycin is probably causing a severe reduction of ATP pools in the embryo. Regardless, a significant amount of transport remains which is apparently driven by energy derived from glycolysis. This is clearly demonstrated by the iodoacetate and oligomytin combination that produces the greatest inhibition of uptake in early blastocysts of all inhibitors studied. These data suggest
196
DEVELOPMENTAL
that both glycolysis and respiration supply energy for methonine uptake in early blastocysts. Effect of Na+ Concentration, K+-Free Media and Ouabain on the Uptake of Amino Acids
The Na+ and K+ gradients have been proposed as possible sources of energy for amino acid transport in other experimental systems (Vidaver, 1964a, b, c; Schultz and Curran, 1970). In mouse embryos, the transport of methionine is Na+ independent at the late four-cell stage, partially Na+ dependent in late morulae, and fully Na+ dependent in early blastocysts (Borland and Tasca, 1974). The effects of Na+- and K+-depleted media and of ouabain, a Na+-K+ ATPase inhibitor (Skou, 1965), on amino acid transport in preimplantation mouse embryos were, therefore, investigated. For late morulae, uptake of methionine in 25 mM Na+ is 50% of uptake in 141 mM Na+, while [3H]phenylalanine uptake in 83 and 25 mM Na+ at the morula stage is not Na+ dependent (Table 2). In contrast, the uptake of both 1.25 x 1O-6 M methionine and of 0.94 x 10eE M phenylalanine in early blastocysts is strongly inhibited by TABLE EFFECT OF NA+
DEPLETION
Labeled amino acid and concentration (M)
[3H]methionine (1.25 x lOme)
[*H]phenylalanine (0.94 x 10-q
VOLUME 46,1975
BIOLOGY
the depletion of Na+ from the medium (Table 2). In order to characterize further the Na+ dependency for methionine transport, early blastocyst-stage mouse embryos were incubated for 15 min in 1.25 x 10e6, 2.50 x 10ee, and 7.50 x 10e6 M [9H Jmethionine, while varying the Na+ concentration (141, 83, and 25 mM Na+). Double reciprocal Lineweaver-Burk plots constructed from these 15-min uptake values show a competitive type of inhibition (Fig. 1). The K, for methionine uptake increases as the Na+ concentration is decreased in the extracellular medium. The role of cation gradients in maintaining methionine transport was investigated by measuring the uptake of methionine in K+-free medium, and in ouabain ( 10msM). One- and two-hour simultaneous incubation of early blastocysts in K+-free FMOCG with labeled methionine do not decrease methionine uptake (Table 3). Incorporation is inhibited to 56% of the control value in 2 hr in K+-free medium. Amino acid transport in early blastocysts may be relatively insensitive to ouabain and to K+-free medium, owing to storage of cations in the blastocoele and to ionic fluxes across the blastocoelic surface 2
ON THE UPTAKE OF [aH]M~~~~~~~ AND [JH]~~~~~~~~~ LATE MORULA, AND BLASTOCYST STAGE MOUSE EMBRYOSO
IN LATE
FOUR-CELL,
Late four-cell stage TCA-soluble fraction (dpm/embryo/hr)
Late morula stage TCA-soluble fraction (dpm/embryo/hr)
Early blastocysts TCA-soluble fraction (dpm/embryoihr)
141
137.4 + 14.6 (100)
173.5 zt 9.6 (100)
539.3 f 47.1(100)
83 25 141 83 25
143.6 f 18.3 (105) 124.9 * 11.8 (91) -
Na+ concentration (mM)
-
88.3 * 3.7 (51) 171.0 * 13.0 (100) 194.0 * 24.0 (113) 169.5 + 1.8 (99)
364.0 106.9 270.3 199.0 146.8
zt 14.9 (67) f 16.2 (20) zt 5.57 (100) f 11.1 (74) zt 22.4 (54)
“Thirty late four-cell embryos, 20 late morulae, or 20 early blastocysts were used per experiment. The embryos were labeled in Na+-depleted or normal FMOCG for 1 hr at 37°C and were then washed and TCA-extracted. Choline chloride replaced NaCl in the Nat-depleted medium in order to maintain osmoticity. Values are the mean * the standard error of the mean. The values in parentheses are the percentages of the
control (141 mM Na+) uptake values.
BORLAND AND TASCA
‘IS
(X
IO+
Methionine
M )
FIG. 1. Na+-dependency kinetics for [sH]methionine uptake in early blastocysts; Lineweaver-Burk plot. Twenty early blastocysts were used for each experiment. The embryos were labeled with [‘Hlmethionine (abscissa, from 1.25 x lo-“7.50 x 10e6 M, expressed as l/S) in either normal FMOCG (141 mM Na+) or Na+-depleted media (83, 23 mM) for 15 min at 37°C. The TCA-soluble fraction (u) was obtained as described in Materials and Methods and is expressed on the graph as l/u in dpm of [‘Hlmethionine/embryo/l5 min (ordinate). Values are the averages of at least three determinations.
of the trophoblastic cells lining the blastocoele. Late morula stage embryo were, therefore, exposed to these identical conditions. One-hour incubations of late morula stage embryos with [SH]methionine in ouabain (lOms M) and in K+-free FMOCG markedly inhibit uptake and incorporation of labeled methionine (Table 3). However, [*H]phenylalanine uptake, which is Na+ independent in the late morula, but Na+ dependent in the early blastocyst, is not inhibited by l-hr incubations with ouabain (lo-* M) or in K+-free FMOCG at the late morula stage (Table 3). DISCUSSION
The preincubation-type experiments employed in these studies with inhibitors indicate that transport of methionine in late four-cell embryos is inhibited by oli-
Uptake in Mouse Embryos
197
gomycin but unaffected by either iodoacetate or ouabain. Uptake of methionine in early blastocyst stage embryos is inhibited by either oligomycin, ouabain or iodoacetate and is unaffected by DNP (lo-’ M) or KCN (1O-2 M). The inhibition of methionine uptake by oligomycin (Table 1) in late four-cell and early blastocyst-stage embryos is probably due to oligomycin’s known effectiveness in inhibiting respiration when coupled to phosphorylation (Lardy et al., 1964). Oligomycin has been reported to inhibit directly transport adenosine triphosphatase (ATPase) in cell membranes by acting like ouabain in blocking ATP hydrolysis (Whittam et al., 1964; Blake et al., 1967). The fact that oligomycin inhibits methionine transport to the same degree (45%) in late four-cell and early blastocyst-stage embryos, even though methionine transport is not linked to Na+ at the late four-cell stage, suggests that oligomycin is acting primarily as an inhibitor of mitochondrial transphosphorylation and respiration. The absolute inhibition of methionine transport is, however, greater in the early blastocysts than in the late four-cell embryo. It is, therefore, impossible to eliminate the possibility of simultaneous effects of oligomytin on respiration and on transport ATPase in the cell membrane of the early blastocyst. KCN (lo-* M), an inhibitor of cytochrome oxidase, and dinitrophenol (lo-’ M), an uncoupler of oxidative phosphorylation, do not inhibit uptake or incorporation (unpublished) of methionine in early blastocysts. The uptake of methionine is, in fact, significantly stimulated by lo-’ M DNP in four-cell embryos. The cause of the DNP-induced stimulation is not known. The most direct interaction between respiration and transport is flux coupled to the flow of electrons through the respiratory chain (Conway, 1963). A proposal of this type of coupling could explain the stimulation of uptake, but such a mechanism for
198
DEVELOPMENTALBIOLIXY TABLE
VOLUME 46, 1975 3
EFFECT OF K+-FREE MEDIUM AND OF OUABAIN ON [3H]M~~~r~~~~~ AND [SH]P~~~~~~~~~~~~ UFTAKE AND INCORPORATIONIN EARLY BLASTOCYSTSAND LATE MORULA-STAGE EMBRYOS IN THE MOUSE” Treatment
Dpm [SH]methionine/early TCA-soluble fraction
FMOCG (141 mM Na+) K+-Free FMOCG FMOCG (141 mM Na+) FMOCG + Ouabain (lObaM) K+-Free FMOCG
Percent of control
544.8 zt 35.7 553.3 + 140.2
100 102
blastocyst/l
TCA-insoluble fraction
hr
Percent of control
163.4 zt 21.2 151.9 + 23.2
100 93
23.1 21.5
hr 100 80 56
28.4 33.2 18.5
100 68 69
25.9 30.3 28.0
hr loo 109 117
17.4 17.2 13.3
Dpm [*H]methionine/early blastocyst/2 1504.0 f 222.7 109 598.0 h 25.5 942.3 i 98.5 69 468.7 * 21.0 1462.0 zt 197.3 97 331.0 * 13.4
FMOCG (141 mM Na+) FMOCG + Ouabain (lo-‘M) K+-Free FMOCG
238.6 * 1.27 131.3 * 2.36 149.3 * 0.4
Dpm [‘H]methionine/late morula/l .lOO 83.8 + 5.6 55 56.9 zt 2.9 63 58.1 + 8.8
FMOCG (141 mM Na+) FMOCG + Ouabain (10e9M) K+-Free FMOCG
216.0 i 14.9 237.5 zt 9.5 254.0 zt 0.7
Dpm [SH]phenylalanine/Iate morula/l 100 45.5 i 8.1 110 49.5 * 1.8 118 39.0 f 0.0
Incorporation mJ)
hr
a Thirty late morulae or 20 early blastocysts were incubated for the times shown in the presence of 1.25 x W6 M [SH]methionine or 0.94 x lo-# M [SH]phenylalanine. The values are the mean + the standard error of the mean. The K+-free medium was prepared by modifying FMOCG as follows: Choline chloride equimolarly replaced KCI (4.78 rnA4) and NaH,PO, replaced KH,PO, (1.19 mm. Percentage incorporation values were determined by dividing the isotope incorporation values (in dpm/embryo/time) by the sum of the uptake value and the incorporation value and multiplying this fraction by 100.
transport across the cell membrane of eucaryotic cells is not widely accepted. KCN and DNP may either be impermeable and/or ineffective in sufficiently decreasing ATP pools to inhibit transport at the concentrations used. The inhibition of methionine uptake by iodoacetate in early blastocysts but not late four-cell embryos is consistent with the fact that glucose cannot be utilized as an energy substrate for development prior to the eight-cell stage (Biggers, 1971). lodoacetate, a sulphydryl agent that blocks glyceraldehyde-P-dehydrogenase and the glycolytic pathway, significantly inhibits uptake of methionine at the blastocyst stage. Late four-cell embryos that lack a functioning glycolytic pathway (Biggers, 1971) are not affected by iodoacetate. Determinations of the ATP to ADP ratios in preimplantation mouse embryos (Quinn
and Wales, 1973) have indicated that there is a gradual decline in the ratio from the two-cell stage to the blastocyst stage and a decline in the total ATP content per embryo. The regulation of glycolysis has been suggested to occur by inhibition of phosphofructokinase by high ATP levels (Quinn and Wales, 1973) and by high ATP and citrate levels (Barbehenn et al., 1974). The effect of ouabain and cation depletion were performed in order to elucidate the role of cation gradients in the methionine transport process in the early blastocyst. Double reciprocal Lineweaver-Burk plots of methionine uptake with various Na+-depleted media indicate a competitive type of inhibition. The K, for methionine is decreased as the Na+ concentration is increased in the extracellular medium, possibly indicating a lower carrier affinity for methionine in Na+-depleted media.
BORLAND AND TASCA
Methionine
Ouabain (lOma M) and K+-free medium have no effect on methionine uptake in early blastocysts in l- and 2-hr simultaneous incubations, respectively (Table 3). K+-free medium inhibits incorporation to 56% in 2 hr. This inhibition may be due to inhibition of peptidyl translocation (Pestka, 1971) and is evidence that KC-free medium is probably affecting the intracellular potassium levels. Ouabain (10m3 M) inhibits uptake more than incorporation in the early blastocyst in 2 hr. The high concentration of ouabain used to produce an effect suggests either 1) that uptake is only indirectly linked to the K+ and Na+ gradients, 2) that the Na+-KC ATPase (the sodium pump) is not easily accessible to ouabain, and/or 3) that the Na+ and K+ gradients are difficult to perturb in the early blastocyst. The relative insensitivity of methionine transport in the early blastocyst may be unique due to storage of cations in the blastocoele and to ionic transport across the blastocoelic surface of the trophoblastic cells lining the blastocoele. Na+ and Cl- have been shown to be actively transported into the blastocoelic cavity of rabbit blastocysts (Cross, 1973; Smith, 1970). If the relative insensitivity of methionine transport in early blastocysts is unique owing to those properties mentioned above, Na+-dependent methionine transport in the late morula, which lacks a blastocoelic cavity, should be more sensitive to K+-free medium and to ouabain than is transport in the early blastocyst. This hypothesis is confirmed by the fact that 1-hr incubations of late morulae in ouabain ( 1O-3 M) and in K +-free medium markedly inhibit uptake of [3H]methionine (Table 3). These effects of K+-free medium and of ouabain on the transport of methionine in the late morula are not due to nonspecific toxicity of these treatments at the late morula stage as evident by the lack of effect of these treatments on Na+-independent phenylalanine transport at this stage (Table 3).
Uptake in Mouse Embryos
199
Since ouabain acts on the exterior of the cell membrane, these changes in sensitivity to it are not due to changes in the trophoblast cells’ permeability to the drug. The location of the sodium pump on blastocoelic surface of the trophoblastic cells lining the mouse blastocoele and the storage of cations in the blastocoele could explain the differences in the sensitivity of methionine transport between the late morula and early blastocyst stages to ouabain and to K+-free medium. Sodium pumps in this location may not be accessible to ouabain or to the effects of K+-free medium. The data suggest that the role of the cations for methionine transport in preimplantation mouse embryos may be similar to their role in glycine transport in Ehrlich ascites tumor cells as previously proposed (Lin and Johnstone, 1971; Johnstone, 1972). The linear dependency of methionine and leucine transport upon the extracellular Na+ concentration in early blastocysts (Borland and Tasca, 1974), the role of Na+ in decreasing the K, of methionine transport, and the effect of Na+ depletion in 15 min at the early blastocyst state are consistent with Lin and Johnstone’s (1971) hypothesis that extracellular Na+ acts directly at the cell membrane to increase the affinity of the amino acid carrier for methionine. The lack of effect of K+-free medium, the relatively slight effect of ouabain (even at the excessively large concentration of 10e3 M) and the lack of effect of Na+depleted medium on methionine efflux in early blastocysts (Borland, 1974) suggest that the Na+ gradient is either 1) not critical in driving methionine transport or 2) very difficult to perturb in the early blastocyst. Accumulation may proceed normally as long as sufficient Na+ is present externally and, presumably, if sufficient K+ is present internally. The early blastocyst may be less sensitive to both ouabain and K+-free medium than is the late morula owing to larger intraembryonic pools of K+ in the early blastocyst, which
200
DEVELOPMENTAL.
BIOLOGY
enable the methionine to dissociate from the carrier on the inside of the cell membrane. Calculations based upon the rate of accumulation of blastocoelic fluid in the rabbit blastocoele clearly show that large amounts of K+, Na+ and Cl- are accumulated in the blastocoelic fluid (Biggers, 1972). A large intraembryonic pool of K + in the early mouse blastocyst may also be associated with the blastocoele. In the early blastocysts, the sodium pump may be localized on the blastocoelic surface of the trophoblastic cells lining the blastocoele and may possibly maintain the intracellular K + concentration by pumping K+ into the intracellular space from the blastocoele and Na+ into the blastocoele from the intracellular space. Therefore, the trophoblast cells of the blastocyst may have ion-transport properties similar to other epithelial cell layers (Herrera, 1966; Enders, 1971), which are affected by ouabain on the serosal surface and not on the mucosal surface of the layer, while blastomeres of the late morula behave more like a cell suspension lacking this polarity and with more accessible Na+-K+ ATPases. Gamow and Daniel (1970) have also emphasized the structural and functional analogy between mammalian blastocysts and epithelial tissues. Additional evidence for trophoblast polarity and Nat-K+ ATPase localization has come from recent studies which indicate that mouse blastocysts collapsed with cytochalasin B cannot reexpand in the absence of Na+ or K+ and in the presence of ouabain (DiZio and Tasca, 1974). Na+depleted medium, K+-free medium and ouabain do not cause collapse of early mouse blastocysts in culture but do block this reexpansion process. Blastocysts collapsed with cytochalasin B and incubated in Na+-depleted or K+-free medium or in the presence of ouabain can reexpand into normal appearing blastocysts when subsequently placed into control medium. Although the exact effect of cytochalasin B
VOLUME 46,1975
on mouse blastocysts is not known, these findings support the hypothesis presented in this paper that ouabain-sensitive sites may be localized on the blastocoelic surface of the blastocyst. In summary, we suggest here that the role of Nat in the Na+-dependent methionine transport system in mouse blastocysts is to increase the affinity of methionine for a membrane-bound carrier. Respiration provides energy for methionine transport in late four-cell embryos, and respiration and glycolysis provide energy for methionine transport in early blastocysts. In addition, these studies provide support for the idea that blastocysts are structurally and functionally analogous to epithelial tissues and may possess Na+-K+ ATPases localized on their blastocoelic surfaces. REFERENCES E. K., WALES, R. G., and LOWRY, D. H. (1974). The explanation for the blockade of glycolysis in early mouse embryos. F’roc. Nat. Acad. Sci. USA 71, 1056-1060. BIGGERS, J. D. (1971). Metabolism of mouse embryos. J. Reprod. Fert. 14, Suppl., 41-45. BIGGERS, J. D. (1972) Mammalian blastocyst and amnion formation. ln “The Water Metabolism of the Fetus” (A. C. Barnes and A. E. Seeds, eds.), pp. l-31. C. C Thomas, Springfield, IL. BLAKE, A., LEADER, D. P., and WH~AM, R. (1967). Physical and chemical reactions of phosphates in red cell membranes in relation to active transport. J. Physiol. 193, 467-479. BORLAND, R. M. (1974). Uptake and incorporation of neutral amino acids in preimplantation mouse embryos: activation of a Na+-dependent amino acid transport system. Ph.D. thesis, University of Delaware, Newark, DE. BORLAND, R. M., and TASCA, R. J. (1974). Activation of a Na+-dependent amino acid transport system in preimplantation mouse embryos. Develop. Biol. 36, 169-182. BRINSTER, R. L. (1963). A method for in oitro cultivation of mouse ova from two-cell to blastocyst. Exp. Cell Res. 32, 205-208. BRINSTER, R. L. (1965). Studies on the development of mouse embryos in uitro. IV. Interaction of energy sources. J. Reprod. Fert. 10, 227-240. BRINSTER, R. L. (1971). Uptake and incorporation of amino acids by the preimplantation mouse embryo. J. Reprod. Fert. 27, 329-338. BARBEHENN,
BORLAND AND TASCA
Methionine
CONWAY, E. J. (1963). Significance of various factors including lactic dehydrogenase on the active transport of sodium ions in skeletal muscle. Nature (London) 198, 760-763; CROSS, M. H. (1973). Active sodium and chloride transport across the rabbit blastocoele wall. Biol. Reprod. 8. 566-575. DIZIO, S. M., and TASCA, R. J. (1974). Ion-dependent, ouabain-sensitive reexpansion of mouse blastocysts collapsed with cytochalasin B. Abstracts, 14th Annual Meeting, American Society for Cell Biology, San Diego, CA. EDWARDS, R. G., and GATES, A. H. (1959). Timing of the stages of the maturation divisions, ovulation, fertilization and the first cleavage of eggs of adult mice treated with gonadotrophin. J. Endocrinol. 18, 292-304. ENDERS, A. C. (1971). The fine structure of the blastocyst. In “The Biology of the Blastocyst” (R. J. Blandau, ed.) pp. 72-95. University of Chicago Press, Chicago, London. EPSTEIN, C. J., and SMITH, S. A. (1973). Amino acid uptake and protein synthesis in preimplantation mouse embryos. Develop. Biol. 33, 171-184. GAMOW, E., and DANIEL, J. C. (1970). Fluid transport in the rabbit blastocyst. Wilhelm Roux Arch. Enttvickslungmech. Organismen 164, 261-278. GINSBERG, L., and HILLMAN, N. (1973). ATP metabolism in cleavage-staged mouse embryos. J. Embryol. Exp. Morphol. 30, 267-282. HERRERA, F. C. (1966). Action of ouabain on sodium transport in the toad urinary bladder. Amer. J. Physiol. 210, 980-986. HILLMAN, N., and TASCA, R. J. (1969). Ultrastructural and autoradiographic studies of mouse cleavage stages. Amer. J. Anat. 126, 151-174. JOHNSTONE, R. M. (1972). Glycine accumulation in the absence of Na+ and K+ gradients in Ehrlich ascites cells. Biochim. Biophys. Acta 282,366-373. KIMMICH, G. A., and RANDLES, J. (1973a). Effect of Na+ and K+ gradients on accumulation of sugars by isolated intestinal epithelial cells. J. Membrane Biol. 12, 23-46. KIMMICH, G. A., and RANDLES, J. (1973b). Interaction between Na+-dependent transport systems for sugars and amino acids. Evidence against a role for the sodium gradient. J. Membrane Biol. 12, 47-68. LARDY, H. A., CONNELLY, J. L., and JOHNSON, D. (1964). Antibiotics as tools for metabolic studies. II. Inhibitors of phosphoryl transfer in mitochondria by oligomycin and aurovertin. Biochemistry 3, 1961-1968. LIN, K. T., and JOHNSTONE, R. M. (1971). Active
Uptake in Mouse Embryos
201
transport of glycine by mouse pancreas: Evidence against the Na+-gradient hypothesis. Biochim. Biophys. Acta 249, 144-158. MILLS, R. M., and BREWER, R. L. (1967). Oxygen consumption of preimplantation mouse embryos. Exp. Cell Res. 47, 337-344. Ozus, C. B., and STERN, S. (1973). Glycogen levels of preimplantation mouse embryos developing in vitro. Biol. Reprod. 8, 467-472. PESTKA, S. (1971). Inhibitors of ribosome functions. Ann. Rev. Biochem. 40, 697-710. QUINN, P., and WALES, R. G. (1971). Adenosine triphosphate content of preimplantation mouse embryos. J. Reprod. Fert. 25, 133-135. QUINN, P., and WALES, R. G. (1973). The effect of culture in vitro on the levels of adenosine triphosphate in preimplantation mouse embryos. J. Reprod. Fert. 32, 231-241. SCHULTZ, S. G., and CURRAN, P. F. (1970) Coupled transport of sodium and organic solutes. Physiol. Rev. 50, 637-718. SKOU, J. C. (1965). Enzymatic basis for active transport of Na+ and K+ across cell membrane. Physiol. Rev. 45, 596-617. SMITH, M. S. (1970). Active transport in the rabbit blastocyst. Experientia 26, 736-737. STERN, S., BIGGERS, J. D., and ANDERSON, E. (1971). Mitochondria and early development of the mouse. J. Exp. Zool. 176, 179-192. TASCA, R. J., and HILLMAN, N. (1970). Effects of actinomycin D and cycloheximide on RNA and protein synthesis in cleavage stage mouse embryos. Nature (London) 225, 1022-1025. TUCKER, A. M., and KIMMICH, G. A. (1973) Characteristics of amino acid accumulation by isolated intestinal epithelial cells. J. Membrane Biol. 12, l-22. VIDAVER, G. A. (1964a). Transport of glycine by pigeon red cells. Biochemistry 3, 662-667. VIDAVER, G. A. (1964b). Some tests of the hypothesis that the sodium-gradient furnishes the energy for glycine-active transport by pigeon red cells. Biochemistry 3, 803-808. VIDAVER, G. A. (1964~). Glycine transport by hemolyzed and restored pigeon red cells. Biochemistry 3, 795-799. WALES, R. G. (1970). Effects of ions on the development of the preimplantation mouse embryo in vitro. Aust. J. Biol. Sci. 23, 421-429. WHI’ITAM, R., WHEELER, K. P., and BLAKE, A. (1964). Oligomycin and active transport reactions in cell membranes. Nature (London) 203, 720-724.
Na+-Dependent
Amino
Acid Transport
Mouse II. Metabolic
Inhibitors
in Preimplantation
Embryos
and Nature
of the Cation
Requirement
RAYMOND M. BORLAND’ AND RICHARD J. TASCA~ Department
of Biological
Sciences, University Accepted
of Delaware,
Newark,
Delaware
19711
May 7, 1975
Experiments were conducted in order to determine the energy source and nature of the cation dependency of [aH]methionine transport in preimplantation mouse embryos. The energy source of methionine transport was studied at the late four-cell and early blastocyst stages. The embryos, raised in vitro, were incubated for 1 hr in inhibitor(s) of energy metabolism and then transferred for 1 hr to medium that contained inhibitor(s) and *H-methionine. These inhibitor studies suggest that respiration and glycolysis are needed to maintain uptake of methionine in early blastocysts. Late four-cell embryos seem to utilize respiration alone for transport. The cation dependency of methionine transport was studied at the late morula and early blastocyst stages. The kinetics of methionine uptake by early blastocysts in Na+-depleted media indicate a competitive type of inhibition. The uptake of methionine by early blastocysts is relatively resistant to ouabain and unaffected by K+-free medium. In contrast, methionine uptake by late morula-stage embryos is markedly inhibited by ouabain and K+-free medium in 1 hr. These results suggest that 1) Na+ serves to increase the affinity of methionine for the carrier in early blastocysts, 2) the cation gradients do not supply a major fraction of the energy required for methionine transport, and/or the gradients are difficult to perturb once the blastocyst has formed, and 3) putative Na+ pumps may be localized on the blastocoelic surface of the blastocysts. INTRODUCTION
Recent studies have shown that the permeability of preimplantation stage mouse embryos to exogenously supplied amino acids increases at the eight-cell, morula and blastocyst stages (Tasca and Hillman, 1970; Brinster, 1971; Epstein and Smith, 1973). It is also known that late four-cell embryos and early blastocysts can concentrate leucine (Epstein and Smith, 1973) and methionine (Borland and Tasca, 1974) and transport methionine and leutine by processes that exhibit saturable, Michaelis-Menten-type kinetics (Borland and Tasca, 1974). Additional experiments demonstrated amino acid competition for ‘Present address: Department of Physiology and Laboratory of Human Reproduction and Reproductive Biology, Harvard Medical School, 45 Shattuck Street, Boston, MA 02115. ‘To whom requests for reprints should he addressed.
transport and temperature dependency of amino acid transport which are also characteristic of carrier-mediated, active transport systems (Epstein and Smith, 1973; Borland and Tasca, 1974). The transport of leucine and methionine at the late four-cell stage is Na+ independent, whereas transport of these two amino acids is completely Na+ dependent at the early blastocyst stage (Borland and Tasca, 1974). In other experimental systems, Na+ and K+ gradients have been proposed as sources of energy for amino acid transport (Vidaver, 1964a, b, c; review, Schultz and Curran, 1970). Other studies argue that cellular energy metabolism directly provides the major source of energy for amino acid transport (Tucker and Kimmich, 1973; Kimmich and Randles, 1973a, b; Lin and Johnstone, 1971). The present studies were undertaken in order to determine the source of energy for 192
Copyright 0 1975 by AcademicPress,Inc. AU rights of reproductionin any form reserved.
BORLAND
AND TASCA
Methionine
amino acid transport in preimplantation mouse embryos and to further characterize the Na+ dependency of transport. Therefore, experiments were designed to determine the effects upon amino acid transport at the late four-cell, morula and blastocyst stages of 1) inhibitors of energy metabolism and 2) Na+ concentration, K+-free media, and ouabain. MATERIALS
Superovulation
AND
METHODS
and
embryo
culture.
Eight- to twelve-week-old random-bred Swiss female mice (Flow Laboratory and Charles Rver Breeding Laboratories) were superovulated with 10 IU of pregnant mare serum gonadotropin (Gestyl, Organon, Inc.) given ip, followed 44 hr later by 10 IU of human chorionic gonadotropin, ip (Pregnyl, Organon, Inc.) (Edwards and Gates, 1959). The females were mated following the second injection, and those with vaginal plugs were selected as pregnant 21-23 hr later. The pregnant females were sacrificed by cervical dislocation, and the twocell embryos collected from the oviducts on the day following vaginal plug observation. The embryos were raised in vitro according to the method of Brinster (1963) in Brinster’s medium for ovum culture (BMOC) (Brinster, 1965) under light-weight paraffin oil (Fisher 125/135). Late four-cell embryos were used for experiments after 14-20 hr in vitro. Morulae and early blastocysts had been in vitro for 46-52 hr by the time of experimentation. Blastocysts at identical stages of development, as measured by the degree of blastocyst expansion, were used for each series of experiments. Early blastocysts contained 20-30 cells as determined by acetic-orcein staining. Labeling procedures. The completely defined medium (FMOCG, Ficoll medium for ovum culture with glucose) used during preincubation and labeling periods consisted of Brinster’s medium for ovum culture (BMOC, Brinster, 1965) but without bovine serum albumin, which was replaced
Uptake in Mouse Embryos
193
by 1 g/liter of Ficoll (Pharmacia), a nonmetabolizable, synthetic, sucrose polymer, and containing 0.1% glucose (Fisher Scientific Co.). In the preincubation inhibitor studies (Table l), late four-cell and early blastocyst-stage embryos were incubated for 1 hr in inhibitor(s) and then transferred for 1 hr to medium which contained inhibitor(s) and [sH]methionine. The experimental culture medium for labeling was prepared by adding [sH]methionine to FMOCG. Commercially prepared L- [methyl-sH]methionine, sp act, 4.02 Ci/mmole, sp act, and L- [3-3H(N)]phenylalanine, 5.39, were obtained from New England Nuclear Corporation. The inhibitors used in this series of studies were purchased from the following companies: Iodoacetic acid (Eastman Organic Chemicals); 2,4dinitrophenol (DNP) (Fisher Scientific Co.); potassium cyanide (KCN) (J. T. Baker Chemical Co., Phillipsburg, NJ); oligomycin (15% oligomycin A and 85% oligomycin B, Sigma Chemical Co.); and strophanthin G (ouabain, Sigma Chemical Co.). Twenty early blastocysts, 20 late morulae, or 30 late four-cell embryos were incubated per experiment. One drop of medium (Vi-Y2 ml) was placed in a 60 x 15-mm tissue culture dish (Falcon Plastics R3002). Embryos to be preincubated and labeled were selected randomly and transferred in lo-20 ~1 of BMOC in a micropipet directly to the drop of experimental medium. Light-weight paraffin oil was not used to cover the drops of experimental media due to the lipid solubility of some of the inhibitors. The embryos were incubated during the experimental treatment in high humidity in 5% CO, and 95% air at 37°C unless stated otherwise. After incubation the embryos were transferred sequentially in a micropipet, as stated above, through four changes of ice-cold (0-4°C) BMOC under light-weight paraffin oil, and immediately frozen in a tissue culture tube in an acetone-solid CO, bath.
194
DEVELOPMENTAL
BIOL~C:Y
The Na+-depleted medium (25 mM Na+) was prepared by modifying BMOC (Brinster, 1965) as follows: Choline chloride (Sigma Chemical Co.) equimolarly replaced NaCl, K-lactate replaced Na-lactate, K-pyruvate replaced Na-pyruvate, 1 mg/ml of Ficoll replaced bovine serum albumin, and the medium contained 0.1% glucose. K-lactate was prepared as follows: 1.82 ml of concentrated lactic acid (m-lactic acid, free acid, Sigma Chemical Co.) was added to 200 ml of double-distilled water, neutralized to pH 7.4 (about 15 ml of 1 N KOH), and added to a final volume of 1 liter of medium. Various concentrations of Na+-containing medium were then obtained by appropriately diluting Na+depleted medium with FMOCG. Thus, normal BMOC contains 5.97 mM K+, the Na+-depleted medium contains 26.97 mM K+. K+ concentrations as high as this do not affect normal embryonic development (Wales, 1970) and are normally present in the mouse oviduct (Roblero, Biggers and Lechene, personal communication). Extraction procedures. The extraction procedures were a modification of the procedures of Brinster (1971). The sample tubes were stored at -20°C until extraction. One milligram of bovine serum albumin and 1 mg of cold carrier amino acid in 20 ~1 of water were added to each sample. The tubes were then freezethawed four times with mild agitation during each thawing to insure complete breakdown of cell structures. One hundred microliters of ice-cold 10% trichloroacetic acid (TCA) was added to each tube, the contents gently mixed, the tubes stored on ice for 10 min, and then centrifuged at 10,000 rpm for 10 min in a Sorvall RC-2 centrifuge (SS-34 rotor). The TCA supernatant fluid was drawn off with a finely drawn Pasteur pipet and saved in a scintillation vial (TCA-soluble fraction). The TCA precipitate was then resuspended in 200 ~1 of 5% TCA, the tube vortexed gently, and then stored on ice for 10 min. The
Vo~m1~46,1975
tubes were again centrifuged at 10,000 rpm in a Sorvall RC-2 and the supernatant fluid removed and pooled with the soluble fraction. The precipitate was dissolved in 100 ~1 of 10% KOH for 30 min and was then transferred to a scintillation vial (TCAinsoluble fraction). Ten milliliters of Cocktail D (100 g of naphthalene, 5 g of 2,5diphenyloxazole in 1 liter of p-dioxane) were added to each vial, and the vials were counted on a Beckman liquid scintillation counter (LS-230). Counting efficiencies (35-40%) were determined by the method of internal standards, and all values are expressed in dpm/embryo/time interval. FtESULTS
Effects
of Inhibitors
on the
Uptake
of
Various inhibitors (oligomycin, 5 x 10ms g/ml; 2,4-dinitrophenol, 1O-s-1O-4 M, and KCN, lo-* M) were used in initial experiments to determine the source(s) of energy for methionine transport in early blastocyst stage mouse embryos. Embryos were incubated for 1 hr simultaneously in FMOCG with [sH]methionine and one of the above inhibitors. Under these conditions, methionine transport was not inhibited. It is possible that the 1 hr incubation does not sufficiently deplete the endogenous energy supply to affect methionine transport. Therefore, a series of preincubation experiments (Table 1) were conducted in which early blastocysts were incubated for 1 hr in inhibitor(s) and then transferred for 1 hr to medium which contains inhibitor(s) and [sH]methionine. The uptake of [sH]methionine (1.25 x 1OT8 M) has been shown to be linear for 1 hr at both the late four-cell and early blastocyst stages (Borland and Tasca, 1974). Culture medium droplets were not placed under oil for these experiments because of the high partition coefficient of some of the inhibitors. In view of the known changes during preimplantation mouse development in energy metabolism (Biggers, 1971), glycogen lev-
BORLAND
AND TAWA
Methionine TABLE
EFFECT OF INHIBITORY
ON [‘HIMETHIONINE
UPTAKE
1 IN FOUR-CELL EMBRYOS AND EARLY BLASTOCYSTS”
Early blastocyst
Late four cell
Treatment
TCA-soluble fraction
Percent of control
(dpm/embryo/hr) Control Ouabain (lo-’ IW) KCN (10-l iI!) Iodoacetic acid (lo-‘Ikf) 2,4-Dinitrophenol (lO-’ M Oligomycin (5 x lo-’ g/ml) Iodoacetic acid (lOma I@) + oligomycin (5 x 10--Bg/ml)
195
Uptake in Mouse Embryos
119.5 * 4.9 138.3 + 17.7 163.5 + 16.6 183.3 zt 16.6” 53.8 + 8.2’ 48.4 + 13.2b
TCA-soluble fraction
Percent of control
(dpm/embryo/hr) 100 116 137 153 45 41
537.1 385.7 566.7 107.0 576.4 240.7 38.8
zt 43.8 + 20.3b zt 8.4 zt 12.7c z+z27.8 f 35.2’ + 6.6’
100 72 106 20 107 45 7
“Thirty late four-cell or 20 early blastocyst-stage mouse embryos were preincubated in FMOCG with inhibitor(s) for 1 hr, and then incubated for an additional 1 hr in FMOCG with inhibitor(s) containing 1.25 x 10ee A4 L- [methyl-3H]methionine. After labeling, the embryos were washed, frozen, TCA-extracted, and the TCA-soluble and -insoluble fractions counted. The values are the mean + the standard error of the mean. The culture media drops were not placed under light paraffin oil in these experiments owing to the high lipid solubilitv of some of the inhibitors. The significance of the differences between samples was determined using Student’s t-test. b Significant at P < 0.01. c Significant at P < 0.001.
els (Ozias and Stern, 1973), ATP content and metabolism (Quinn and Wales, 1971, 1973; Ginsberg and Hillman, 1973), mitochondrial structure (Hillman and Tasca, 1969; Stern et al. 1971) and 0, consumption (Mills and Brinster, 1967), identical experiments were conducted on late fourcell embryos to compare the energetics of methionine transport between the two stages. As shown in Table 1, the uptake of methionine in late four-cell embryos is not significantly affected by ouabain (10m3M), an inhibitor of Na+-K+ ATPase. Iodoacetate (10e3 M), an inhibitor of glyceraldehyde-P-dehydrogenase, and 2,4-dinitrophenol (DNP) (lo-’ M), an uncoupler of oxidative phosphorylation, do not inhibit uptake. In fact, DNP (10-l M) significantly stimulates uptake, while inhibiting incorporation by 60% (unpublished). Oligomycin (5 x lOmeg/ml), an inhibitor of respiration (Lardy et al., 1964), decreases uptake to 45% of the control value. This inhibition was not further decreased by the simultaneous presence of iodoacetate and
oligomycin, suggesting that glycolysis is not an important source of energy for methionine transport at the four-cell stage. In the early blastocyst-stage mouse embryo, ouabain ( 1O-3M) decreases uptake of [3H]methionine to approximately 72% of the control value (Table 1). Iodoacetate (lOma M) markedly decreases uptake (to 20% of control value). KCN (10e2 M), an inhibitor of cytochrome oxidase, and DNP (lo-’ M) do not significantly affect uptake of methionine in early blastocysts. Oligomycin (5 x 1Om8g/ml) markedly inhibits both uptake and incorporation (unpublished) but to different degrees (55 and 95%, respectively). This virtual cessation of incorporation indicates that oligomycin is probably causing a severe reduction of ATP pools in the embryo. Regardless, a significant amount of transport remains which is apparently driven by energy derived from glycolysis. This is clearly demonstrated by the iodoacetate and oligomytin combination that produces the greatest inhibition of uptake in early blastocysts of all inhibitors studied. These data suggest
196
DEVELOPMENTAL
that both glycolysis and respiration supply energy for methonine uptake in early blastocysts. Effect of Na+ Concentration, K+-Free Media and Ouabain on the Uptake of Amino Acids
The Na+ and K+ gradients have been proposed as possible sources of energy for amino acid transport in other experimental systems (Vidaver, 1964a, b, c; Schultz and Curran, 1970). In mouse embryos, the transport of methionine is Na+ independent at the late four-cell stage, partially Na+ dependent in late morulae, and fully Na+ dependent in early blastocysts (Borland and Tasca, 1974). The effects of Na+- and K+-depleted media and of ouabain, a Na+-K+ ATPase inhibitor (Skou, 1965), on amino acid transport in preimplantation mouse embryos were, therefore, investigated. For late morulae, uptake of methionine in 25 mM Na+ is 50% of uptake in 141 mM Na+, while [3H]phenylalanine uptake in 83 and 25 mM Na+ at the morula stage is not Na+ dependent (Table 2). In contrast, the uptake of both 1.25 x 1O-6 M methionine and of 0.94 x 10eE M phenylalanine in early blastocysts is strongly inhibited by TABLE EFFECT OF NA+
DEPLETION
Labeled amino acid and concentration (M)
[3H]methionine (1.25 x lOme)
[*H]phenylalanine (0.94 x 10-q
VOLUME 46,1975
BIOLOGY
the depletion of Na+ from the medium (Table 2). In order to characterize further the Na+ dependency for methionine transport, early blastocyst-stage mouse embryos were incubated for 15 min in 1.25 x 10e6, 2.50 x 10ee, and 7.50 x 10e6 M [9H Jmethionine, while varying the Na+ concentration (141, 83, and 25 mM Na+). Double reciprocal Lineweaver-Burk plots constructed from these 15-min uptake values show a competitive type of inhibition (Fig. 1). The K, for methionine uptake increases as the Na+ concentration is decreased in the extracellular medium. The role of cation gradients in maintaining methionine transport was investigated by measuring the uptake of methionine in K+-free medium, and in ouabain ( 10msM). One- and two-hour simultaneous incubation of early blastocysts in K+-free FMOCG with labeled methionine do not decrease methionine uptake (Table 3). Incorporation is inhibited to 56% of the control value in 2 hr in K+-free medium. Amino acid transport in early blastocysts may be relatively insensitive to ouabain and to K+-free medium, owing to storage of cations in the blastocoele and to ionic fluxes across the blastocoelic surface 2
ON THE UPTAKE OF [aH]M~~~~~~~ AND [JH]~~~~~~~~~ LATE MORULA, AND BLASTOCYST STAGE MOUSE EMBRYOSO
IN LATE
FOUR-CELL,
Late four-cell stage TCA-soluble fraction (dpm/embryo/hr)
Late morula stage TCA-soluble fraction (dpm/embryo/hr)
Early blastocysts TCA-soluble fraction (dpm/embryoihr)
141
137.4 + 14.6 (100)
173.5 zt 9.6 (100)
539.3 f 47.1(100)
83 25 141 83 25
143.6 f 18.3 (105) 124.9 * 11.8 (91) -
Na+ concentration (mM)
-
88.3 * 3.7 (51) 171.0 * 13.0 (100) 194.0 * 24.0 (113) 169.5 + 1.8 (99)
364.0 106.9 270.3 199.0 146.8
zt 14.9 (67) f 16.2 (20) zt 5.57 (100) f 11.1 (74) zt 22.4 (54)
“Thirty late four-cell embryos, 20 late morulae, or 20 early blastocysts were used per experiment. The embryos were labeled in Na+-depleted or normal FMOCG for 1 hr at 37°C and were then washed and TCA-extracted. Choline chloride replaced NaCl in the Nat-depleted medium in order to maintain osmoticity. Values are the mean * the standard error of the mean. The values in parentheses are the percentages of the
control (141 mM Na+) uptake values.
BORLAND AND TASCA
‘IS
(X
IO+
Methionine
M )
FIG. 1. Na+-dependency kinetics for [sH]methionine uptake in early blastocysts; Lineweaver-Burk plot. Twenty early blastocysts were used for each experiment. The embryos were labeled with [‘Hlmethionine (abscissa, from 1.25 x lo-“7.50 x 10e6 M, expressed as l/S) in either normal FMOCG (141 mM Na+) or Na+-depleted media (83, 23 mM) for 15 min at 37°C. The TCA-soluble fraction (u) was obtained as described in Materials and Methods and is expressed on the graph as l/u in dpm of [‘Hlmethionine/embryo/l5 min (ordinate). Values are the averages of at least three determinations.
of the trophoblastic cells lining the blastocoele. Late morula stage embryo were, therefore, exposed to these identical conditions. One-hour incubations of late morula stage embryos with [SH]methionine in ouabain (lOms M) and in K+-free FMOCG markedly inhibit uptake and incorporation of labeled methionine (Table 3). However, [*H]phenylalanine uptake, which is Na+ independent in the late morula, but Na+ dependent in the early blastocyst, is not inhibited by l-hr incubations with ouabain (lo-* M) or in K+-free FMOCG at the late morula stage (Table 3). DISCUSSION
The preincubation-type experiments employed in these studies with inhibitors indicate that transport of methionine in late four-cell embryos is inhibited by oli-
Uptake in Mouse Embryos
197
gomycin but unaffected by either iodoacetate or ouabain. Uptake of methionine in early blastocyst stage embryos is inhibited by either oligomycin, ouabain or iodoacetate and is unaffected by DNP (lo-’ M) or KCN (1O-2 M). The inhibition of methionine uptake by oligomycin (Table 1) in late four-cell and early blastocyst-stage embryos is probably due to oligomycin’s known effectiveness in inhibiting respiration when coupled to phosphorylation (Lardy et al., 1964). Oligomycin has been reported to inhibit directly transport adenosine triphosphatase (ATPase) in cell membranes by acting like ouabain in blocking ATP hydrolysis (Whittam et al., 1964; Blake et al., 1967). The fact that oligomycin inhibits methionine transport to the same degree (45%) in late four-cell and early blastocyst-stage embryos, even though methionine transport is not linked to Na+ at the late four-cell stage, suggests that oligomycin is acting primarily as an inhibitor of mitochondrial transphosphorylation and respiration. The absolute inhibition of methionine transport is, however, greater in the early blastocysts than in the late four-cell embryo. It is, therefore, impossible to eliminate the possibility of simultaneous effects of oligomytin on respiration and on transport ATPase in the cell membrane of the early blastocyst. KCN (lo-* M), an inhibitor of cytochrome oxidase, and dinitrophenol (lo-’ M), an uncoupler of oxidative phosphorylation, do not inhibit uptake or incorporation (unpublished) of methionine in early blastocysts. The uptake of methionine is, in fact, significantly stimulated by lo-’ M DNP in four-cell embryos. The cause of the DNP-induced stimulation is not known. The most direct interaction between respiration and transport is flux coupled to the flow of electrons through the respiratory chain (Conway, 1963). A proposal of this type of coupling could explain the stimulation of uptake, but such a mechanism for
198
DEVELOPMENTALBIOLIXY TABLE
VOLUME 46, 1975 3
EFFECT OF K+-FREE MEDIUM AND OF OUABAIN ON [3H]M~~~r~~~~~ AND [SH]P~~~~~~~~~~~~ UFTAKE AND INCORPORATIONIN EARLY BLASTOCYSTSAND LATE MORULA-STAGE EMBRYOS IN THE MOUSE” Treatment
Dpm [SH]methionine/early TCA-soluble fraction
FMOCG (141 mM Na+) K+-Free FMOCG FMOCG (141 mM Na+) FMOCG + Ouabain (lObaM) K+-Free FMOCG
Percent of control
544.8 zt 35.7 553.3 + 140.2
100 102
blastocyst/l
TCA-insoluble fraction
hr
Percent of control
163.4 zt 21.2 151.9 + 23.2
100 93
23.1 21.5
hr 100 80 56
28.4 33.2 18.5
100 68 69
25.9 30.3 28.0
hr loo 109 117
17.4 17.2 13.3
Dpm [*H]methionine/early blastocyst/2 1504.0 f 222.7 109 598.0 h 25.5 942.3 i 98.5 69 468.7 * 21.0 1462.0 zt 197.3 97 331.0 * 13.4
FMOCG (141 mM Na+) FMOCG + Ouabain (lo-‘M) K+-Free FMOCG
238.6 * 1.27 131.3 * 2.36 149.3 * 0.4
Dpm [‘H]methionine/late morula/l .lOO 83.8 + 5.6 55 56.9 zt 2.9 63 58.1 + 8.8
FMOCG (141 mM Na+) FMOCG + Ouabain (10e9M) K+-Free FMOCG
216.0 i 14.9 237.5 zt 9.5 254.0 zt 0.7
Dpm [SH]phenylalanine/Iate morula/l 100 45.5 i 8.1 110 49.5 * 1.8 118 39.0 f 0.0
Incorporation mJ)
hr
a Thirty late morulae or 20 early blastocysts were incubated for the times shown in the presence of 1.25 x W6 M [SH]methionine or 0.94 x lo-# M [SH]phenylalanine. The values are the mean + the standard error of the mean. The K+-free medium was prepared by modifying FMOCG as follows: Choline chloride equimolarly replaced KCI (4.78 rnA4) and NaH,PO, replaced KH,PO, (1.19 mm. Percentage incorporation values were determined by dividing the isotope incorporation values (in dpm/embryo/time) by the sum of the uptake value and the incorporation value and multiplying this fraction by 100.
transport across the cell membrane of eucaryotic cells is not widely accepted. KCN and DNP may either be impermeable and/or ineffective in sufficiently decreasing ATP pools to inhibit transport at the concentrations used. The inhibition of methionine uptake by iodoacetate in early blastocysts but not late four-cell embryos is consistent with the fact that glucose cannot be utilized as an energy substrate for development prior to the eight-cell stage (Biggers, 1971). lodoacetate, a sulphydryl agent that blocks glyceraldehyde-P-dehydrogenase and the glycolytic pathway, significantly inhibits uptake of methionine at the blastocyst stage. Late four-cell embryos that lack a functioning glycolytic pathway (Biggers, 1971) are not affected by iodoacetate. Determinations of the ATP to ADP ratios in preimplantation mouse embryos (Quinn
and Wales, 1973) have indicated that there is a gradual decline in the ratio from the two-cell stage to the blastocyst stage and a decline in the total ATP content per embryo. The regulation of glycolysis has been suggested to occur by inhibition of phosphofructokinase by high ATP levels (Quinn and Wales, 1973) and by high ATP and citrate levels (Barbehenn et al., 1974). The effect of ouabain and cation depletion were performed in order to elucidate the role of cation gradients in the methionine transport process in the early blastocyst. Double reciprocal Lineweaver-Burk plots of methionine uptake with various Na+-depleted media indicate a competitive type of inhibition. The K, for methionine is decreased as the Na+ concentration is increased in the extracellular medium, possibly indicating a lower carrier affinity for methionine in Na+-depleted media.
BORLAND AND TASCA
Methionine
Ouabain (lOma M) and K+-free medium have no effect on methionine uptake in early blastocysts in l- and 2-hr simultaneous incubations, respectively (Table 3). K+-free medium inhibits incorporation to 56% in 2 hr. This inhibition may be due to inhibition of peptidyl translocation (Pestka, 1971) and is evidence that KC-free medium is probably affecting the intracellular potassium levels. Ouabain (10m3 M) inhibits uptake more than incorporation in the early blastocyst in 2 hr. The high concentration of ouabain used to produce an effect suggests either 1) that uptake is only indirectly linked to the K+ and Na+ gradients, 2) that the Na+-KC ATPase (the sodium pump) is not easily accessible to ouabain, and/or 3) that the Na+ and K+ gradients are difficult to perturb in the early blastocyst. The relative insensitivity of methionine transport in the early blastocyst may be unique due to storage of cations in the blastocoele and to ionic transport across the blastocoelic surface of the trophoblastic cells lining the blastocoele. Na+ and Cl- have been shown to be actively transported into the blastocoelic cavity of rabbit blastocysts (Cross, 1973; Smith, 1970). If the relative insensitivity of methionine transport in early blastocysts is unique owing to those properties mentioned above, Na+-dependent methionine transport in the late morula, which lacks a blastocoelic cavity, should be more sensitive to K+-free medium and to ouabain than is transport in the early blastocyst. This hypothesis is confirmed by the fact that 1-hr incubations of late morulae in ouabain ( 1O-3 M) and in K +-free medium markedly inhibit uptake of [3H]methionine (Table 3). These effects of K+-free medium and of ouabain on the transport of methionine in the late morula are not due to nonspecific toxicity of these treatments at the late morula stage as evident by the lack of effect of these treatments on Na+-independent phenylalanine transport at this stage (Table 3).
Uptake in Mouse Embryos
199
Since ouabain acts on the exterior of the cell membrane, these changes in sensitivity to it are not due to changes in the trophoblast cells’ permeability to the drug. The location of the sodium pump on blastocoelic surface of the trophoblastic cells lining the mouse blastocoele and the storage of cations in the blastocoele could explain the differences in the sensitivity of methionine transport between the late morula and early blastocyst stages to ouabain and to K+-free medium. Sodium pumps in this location may not be accessible to ouabain or to the effects of K+-free medium. The data suggest that the role of the cations for methionine transport in preimplantation mouse embryos may be similar to their role in glycine transport in Ehrlich ascites tumor cells as previously proposed (Lin and Johnstone, 1971; Johnstone, 1972). The linear dependency of methionine and leucine transport upon the extracellular Na+ concentration in early blastocysts (Borland and Tasca, 1974), the role of Na+ in decreasing the K, of methionine transport, and the effect of Na+ depletion in 15 min at the early blastocyst state are consistent with Lin and Johnstone’s (1971) hypothesis that extracellular Na+ acts directly at the cell membrane to increase the affinity of the amino acid carrier for methionine. The lack of effect of K+-free medium, the relatively slight effect of ouabain (even at the excessively large concentration of 10e3 M) and the lack of effect of Na+depleted medium on methionine efflux in early blastocysts (Borland, 1974) suggest that the Na+ gradient is either 1) not critical in driving methionine transport or 2) very difficult to perturb in the early blastocyst. Accumulation may proceed normally as long as sufficient Na+ is present externally and, presumably, if sufficient K+ is present internally. The early blastocyst may be less sensitive to both ouabain and K+-free medium than is the late morula owing to larger intraembryonic pools of K+ in the early blastocyst, which
200
DEVELOPMENTAL.
BIOLOGY
enable the methionine to dissociate from the carrier on the inside of the cell membrane. Calculations based upon the rate of accumulation of blastocoelic fluid in the rabbit blastocoele clearly show that large amounts of K+, Na+ and Cl- are accumulated in the blastocoelic fluid (Biggers, 1972). A large intraembryonic pool of K + in the early mouse blastocyst may also be associated with the blastocoele. In the early blastocysts, the sodium pump may be localized on the blastocoelic surface of the trophoblastic cells lining the blastocoele and may possibly maintain the intracellular K + concentration by pumping K+ into the intracellular space from the blastocoele and Na+ into the blastocoele from the intracellular space. Therefore, the trophoblast cells of the blastocyst may have ion-transport properties similar to other epithelial cell layers (Herrera, 1966; Enders, 1971), which are affected by ouabain on the serosal surface and not on the mucosal surface of the layer, while blastomeres of the late morula behave more like a cell suspension lacking this polarity and with more accessible Na+-K+ ATPases. Gamow and Daniel (1970) have also emphasized the structural and functional analogy between mammalian blastocysts and epithelial tissues. Additional evidence for trophoblast polarity and Nat-K+ ATPase localization has come from recent studies which indicate that mouse blastocysts collapsed with cytochalasin B cannot reexpand in the absence of Na+ or K+ and in the presence of ouabain (DiZio and Tasca, 1974). Na+depleted medium, K+-free medium and ouabain do not cause collapse of early mouse blastocysts in culture but do block this reexpansion process. Blastocysts collapsed with cytochalasin B and incubated in Na+-depleted or K+-free medium or in the presence of ouabain can reexpand into normal appearing blastocysts when subsequently placed into control medium. Although the exact effect of cytochalasin B
VOLUME 46,1975
on mouse blastocysts is not known, these findings support the hypothesis presented in this paper that ouabain-sensitive sites may be localized on the blastocoelic surface of the blastocyst. In summary, we suggest here that the role of Nat in the Na+-dependent methionine transport system in mouse blastocysts is to increase the affinity of methionine for a membrane-bound carrier. Respiration provides energy for methionine transport in late four-cell embryos, and respiration and glycolysis provide energy for methionine transport in early blastocysts. In addition, these studies provide support for the idea that blastocysts are structurally and functionally analogous to epithelial tissues and may possess Na+-K+ ATPases localized on their blastocoelic surfaces. REFERENCES E. K., WALES, R. G., and LOWRY, D. H. (1974). The explanation for the blockade of glycolysis in early mouse embryos. F’roc. Nat. Acad. Sci. USA 71, 1056-1060. BIGGERS, J. D. (1971). Metabolism of mouse embryos. J. Reprod. Fert. 14, Suppl., 41-45. BIGGERS, J. D. (1972) Mammalian blastocyst and amnion formation. ln “The Water Metabolism of the Fetus” (A. C. Barnes and A. E. Seeds, eds.), pp. l-31. C. C Thomas, Springfield, IL. BLAKE, A., LEADER, D. P., and WH~AM, R. (1967). Physical and chemical reactions of phosphates in red cell membranes in relation to active transport. J. Physiol. 193, 467-479. BORLAND, R. M. (1974). Uptake and incorporation of neutral amino acids in preimplantation mouse embryos: activation of a Na+-dependent amino acid transport system. Ph.D. thesis, University of Delaware, Newark, DE. BORLAND, R. M., and TASCA, R. J. (1974). Activation of a Na+-dependent amino acid transport system in preimplantation mouse embryos. Develop. Biol. 36, 169-182. BRINSTER, R. L. (1963). A method for in oitro cultivation of mouse ova from two-cell to blastocyst. Exp. Cell Res. 32, 205-208. BRINSTER, R. L. (1965). Studies on the development of mouse embryos in uitro. IV. Interaction of energy sources. J. Reprod. Fert. 10, 227-240. BRINSTER, R. L. (1971). Uptake and incorporation of amino acids by the preimplantation mouse embryo. J. Reprod. Fert. 27, 329-338. BARBEHENN,
BORLAND AND TASCA
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