Copyright Q 1992 by the Genetics Society of America

Hereditary Lactate Dehydrogenase A-Subunit Deficiency as Cause of Early Postimplantation Death of Homozygotes in Mus musculus

Siegbert Merkle,* Jack Favor,* Jochen Graw,* Sabine Hornhardtt and Walter Pretsch*” *Znstitutfiir Saugetiergenetik and TAbteilungfiir Zellchemie, GSF-Forschungszentrumfur Umwelt und Gesundheit GmbH, 0-8042 Neuherberg, Germany Manuscript received January 22, 199 1 Accepted for publication February 3, 1992

ABSTRACT Two ethylnitrosourea-inducedheterozygous mouse mutants with approximately 58 and 50% of wild-type lactate dehydrogenase (LDH) activity and a 7-ray-induced heterozygous mutant with 50% of wild-type LDH activity in blood, liver and spleen (expressing predominantly the Ldh-1 gene) were recovered in mutagenicity experiments following spermatogonial treatment. Physiological and genetic studies revealed no indications for differences in fertility as well ashematological or otherphysiological traits between heterozygotes of each mutant line and wild types. This suggests that neither the mutations in the heterozygous state per se nor the resulting approximate 42 to 50% LDH deficiency affect metabolism and fitness. Physicochemical and immunological studies clearly demonstrated that the two mutations with 50% deficiency inheterozygotes result from null alleles ofthe Ldh-I structural locus, generating neither enzyme activity nor immunological cross-reacting material. In contrast, the heterozygous mutant with approximately 58% of normal blood LDH activity was shown to be due to a Ldh-I allele creating protein subunits, which in random assortment with wild-type subunits in vivo exhibit a reduced specific activityand furtheralterations of kineticand physicochemical characteristics. All the mutations in the homozygous state were found to be lethal at an early postimplantation stage of embryonic development, probably due to a block of glycolysis with the corresponding loss of the main source of metabolic energy during this ontogenetic stage. The distinct physiological consequences of the total absence of a functioning LDH-A subunit in mice and humans are discussed. The key role regarding the presence as well as developmental pattern of isozymes in estimating the impact of enzyme-activity mutations on the phenotype of an organism is emphasized.

ACTATE dehydrogenase (LDH, L-lactate: NAD+ LDH deficiency with respect to metabolism and phys- L oxidoreductase, EC 1.1.1.27.) catalyzes the re- iology in mouse and man might be particularly impor- versible conversion of lactate to pyruvate and thereby tant since information on the impact of induced or plays an important role bothin anaerobic glycolysis as spontaneous mutations at homologous genetical end- well as in lactateoxidation. In mammals there are points would improve understanding of the genetic three separate structural LDH genes, the expression risks of radiation and chemicals for humans. of which is developmentally regulated and tissue spe- In the present study results of the genetic, physio- cific (LI 1989). logical and biochemical characterizations of three Several electrophoreticLDH variants, as well as LDH-deficient mouse mutants are reported. Regard- cases of complete deficiency of either the A or B less of the natureof the individual mutations, all were subunit have been reportedin humans (VESELL1965; found tobe homozygous lethal at anearly postimplan- KITAMURAet al. 197 1; TANIS, NEELand TORRESDE tation stageof embryonic development.This phenom- ARAUZ 1977;KANNO et al. 1980; MOHRENWEISERand enon might be due to a block in glycolysis and the NOVOTNY1982; MAEKAWA,Su~o and KANNO1986) associated loss of the main source of metabolic energy and other mammals (SHAWand BARTO1963; RAUCH because of a totalor almost total loss of LDH mediated 1968; CATTANACHand PEREZ 1969; BRITTON-DAVI- NADH+ reoxidation. DIAN et al. 1978), including the house mouse Mus musculus (SOARES1977, 1978; BRITTON-DAVIDIANet MATERIALS AND METHODS al. 1978; PRETSCHand CHARLES1980; PRETSCH 1989).However, a detailed characterization of the Animals: Two mouse mutants (LDH 1049 and LDH LDH-deficient mutants of the mouse and a compari- 9546) with approximately 58 and 50%wild-type LDH activ- ity in blood were recovered independently in mutagenesis son with humans suffering from LDH deficiency is experiments, inwhich offspring from ethylnitrosourea still lacking. The comparison of the significance of (ENU)-treated (102/EI X C3H/El)FI hybrid males and un- treated test stock femaleswere screened for enzyme activity ’ Author to whom reprint requests should be sent. variants (CHARLESand PRETSCH1987). A third independent

Genetics 131: 413-421 (June, 1992) 414 S. Merkle et al. mutant (LDH 2534) with 50% of normal blood LDH activity cording to the procedure of RADOLA(1980) using Servalyt was a progeny of an AKR male irradiated with a splitdose 4-9T (Serva, Heidelberg, Germany). Polyacrylamidegel of 3 + 3 Gy y-rays with a 24-hr fractionation interval and electrophoresis (PAGE) and staining of the gels were per- an unexposed test stock female (unpublished). For biochem- formed as described by CHARLESand PRETSCH(1 98 1). ical and physiological characterization 10-week-old animals Immunological properties: Antiserum against commer- of both sexes were used, which were backcrossedat least 10 cial, cytoplasmic pig muscle LDH (Boehringer, Mannheim, generations to the C3H/EI wild-type strain (Flo-F15).After Germany) was raised in rabbits. An aliquot containing ap- weaning, four mice of the same sex were housed per cage proximately 1 mg of protein was mixedwith the same and maintained under constant temperature (22 k 2") with volume of Freund's adjuvant and injected subcutaneously. a fixed 12-hr light/l2-hr dark cycle. Tap water and stand- The procedure was repeated at 2-week intervals for a total ardized diet (Altromin 13 14,Altromin International, Lage, of five injections. Blood samples weredrawn after the final Germany) were provided ad libitum. Based on the conclu- injection via venipuncture. After centrifugation the anti- sions drawn from the results of the present and previous serum was stored at -80". To determine the amount of investigation (PRETSCH1989) themutant alleles of the ENU- LDH-cross-reactingmaterial, Western blot analysis was per- induced mutant lines LDH 1049 and LDH 9546 (CHARLES formed as described by TOWBIN,STAEHELIN and GORD~N and PRETSCH1987) and the radiation-induced mutant line (1979) using a Sartoblot semidry blot apparatus (Sartorius, LDH 2534 (unpublished) were designated Ldh-1"-"2A'e",Ldh- Gottingen, Germany) after sodium dodecyl sulfate (SDS)- ln-rn3X.i~and Ldh-l"""4N'",respectively. The mutations will be PAGE according to LAEMMLI(1970) with 200 rg spleen referred to as LDH-ASN, LDH-A3N and LDH-A4N, re- protein. Protein concentration of the samples was deter- spectively. mined according to LOWRYet al. (1951). The antibodies Genetic and physiological analysis: Genetic and phys- bound to the nitrocellulose sheet were stained according to iological characterization of the mutants including deter- the method of STERNBERGER,CUCULIS and MEYER(1970) mination of the stage of homozygouslethality, sampling and using peroxidase conjugated antisera (Bio-Makor, Rehovot, preparation of blood, erythrocytes and tissues, as well as the Israel). The stained bands were scanned at 546 nm using studies of hematologic and other physiological parameters Elscript 400 densitometer (Hirschmann, Unterhaching, followed essentially the procedures previously described in Germany) and the peaks were integrated. detail (EHLINGet al. 1978; MERKLEand PRETSCH1989). Statistical analysis:In the biochemical and physiological Physicochemicaland kinetical properties: Enzymatic characterization experiments, data from the same number activity was determined using the automatic analyzer ACP of female and male animals were used for the mean and 5040 (Eppendorf, Hamburg, Germany). The standard re- SEM. For statistical comparisons between the different gen- action mixture for measuring LDH activity contained IMI otypes, Student's t-test was used. Data from the homozy- buffer (0.1 M imidazol, pH 7.0,70 mM KCI, 18 mM MgS04), gous-lethality experiments were compared using a nonpar- 8.5 mM pyruvate (Na salt) and 0.28 mM NADH (Nar salt). ametric method (Wilcoxon-rank-sum-test).Differences were Measurement of the Michaelis-Menten constants (X,) for stated as significant for P values less than 0.05. pyruvate and NADH were performed using the standard assay for LDHwith varying concentrations of pyruvate RESULTS (0.05; 0.1; 0.2; 0.4; 2 mM) or NADH (0.007; 0.014; 0.028; 0.042; 0.28 mM), respectively. The apparent K, was esti- Genetic characterization and stageof homozygous mated by a least-squares regression fit. lethality in utero: Table 1 summarizes the results of The substrate specifity of LDH was tested by comparing the geneticstudies. Backcrossing heterozygous ani- the activity of the enzyme with 0.7 mM glyoxalate as sub- strate with that obtained in an assay containing 0.3 mM mals from the three independent Ldh-l mutant lines pyruvate as substrate (MOHRENWEISERand NOVOTNY1982). with wild-type C3H/El animals yielded normal litter Noncompetitive inhibition of LDH by oxalate (NISSEL- sizes withhomozygous wild-type andheterozygous BAUM, PACKERand BODANSKY1964) was studied in the mutant offspring in a ratio of approximately 1: 1. In standard assay containing 0.1 mM pyruvate and 0.7 mM contrast, intercrossing heterozygotes of each mutant oxalate (Na salt).The inhibition of LDH by high concentra- tions of pyruvate was measured in the standard assay and is line resulted in a reduction of litter size. Only wild givenas percent activity with 8.5 or 20 mM pyruvate as types and heterozygotes in an approximate 1:2 ratio substrate compared tothe maximalactivity with 2 mM wereobtained in the mutant lines LDH-A2Nand pyruvate as substrate. All determinations of kinetic and LDH-A4N. In the LDH-A3N mutant line, however, inhibitory properties were performed in erythrocyte lysates beside 236 mice with normal and 469 mice with half at 25O. LDH activity inthe assays of homozygous wild types were adjusted to that of heterozygous mutants by diluting of normal LDHactivity 4 animals with approximately the lysates. 1% residual LDH activity were recovered. Since the Heat stability of erythrocyte LDH was determined by latterdied within a fewweeks afterbirth, it was incubating erythrocyte lysate at 60 and 62 O . The concentra- impossible to genetically determine their genotype. tion of hemoglobin in the lysates was approximately 0.0 15 Theprofound reduction of LDH activity andthe g/ml. At 5-min time intervals aliquots were taken and chilled immediately with ice-cold 0.1 M IMI buffer. After sedimen- concomitant decreaseof hemoglobin concentration in tation of precipitated hemogtobin, residual LDH activity blood (9.9 f. 0.5 g Hb/100 ml 'us. 15.3 k 0.6 g Hb/ was assayed as described above. 100 ml of wild types and heterozygotes) and thevisible A Tris-glycine-phosphate buffer system (BEUTLER,MA- reduction of bodysize suggest that these animals were THAI and SMITH1968) with substrate concentrations of the homozygous mutants. In this context it mustbe noted standard assay was used to determine the pH dependence of LDH. that three out of these 4 animals occurred in inter- Isoelectric focusing was carried out on ultrathin-layer crosses of F1 heterozygotesand the fourth case in polyacrylamide gels with a final pH range of 3.0-9.0 ac- intercrosses of F5 animals. Among approximately 300 D eficiency in MiceLDH-A Subunitin Deficiency 415

TABLE 1 Genetic characterizationof three LDH-A deficiency mutants in mice

Reduction of Cross Ldh,l Mean litter +/+ +/- -/- Ratio litter size genotype (n)size i SD (n) (n) +/+:+/- +/+ x +/+ 6.5130 f 1.9 LDH-A2N +/- x +/+ 6.5 f455 1.9 419 1:1.09 +/- x +/- 5.3 f191 1.4 108 0 1:1.77 19 LDH-A3N +/- x +/+ 6.8 k 1.7 308 312 1:1.01 +/- x +/- 5.4 f 2.0 236 469 4 1: 1.99 21 LDH-A4N +/- x +/+ 6.8 f 1.9 136 132 1:0.97 +I- x +/- 5.2 *104 2.3 48 0 1:2.17 24 a +/+ = wild type; +/- = heterozygous mutant; -1- = homozygous mutant. Reduction of litter size of intercrosses compared to backcrosses is calculated as follows: (1 - litter size of intercrosses:litter size of backcrosses) X 100. offspring of intercrosses from later generations (F6- 0.3 g/liter), number of red blood cells (WTV = 8.4 f FI5),so far no additional animals with this phenotype 0.2 X lo'* cells/liter), mean cellular fragility (WTV = could be identified. 0.497 f 0.007%NaCl) and glucose consumption of The reduction in litter size together with the ab- blood (WTV = 2.9 f 0.4 mg/g Hb/hr). Other phys- sence or thesignificantly reduced number of animals iological traits such as plasma glucose (WTV = 1.35 with a third phenotype in intercrosses suggest that f 0.18 g/liter), body weight (WTV = 22.8 f 0.6 g) mutant homozygotes are lethal. Indeed, the genetic and organo-somatic indices (organ weight X 1OO/body testing of 20 randomly chosen offspring from inter- weight) of liver (WTV = 5.65 f 0.20 g/100 g body crosses with about half of normal LDH activity indi- weight), lung(WTV = 0.58 f 0.02g/100 g body cated heterozygosity for these animals. Therefore, the weight), kidney (WTV = 1.36 f 0.04 g/100 g body possibility can beexcluded that the mutations are weight), spleen (WTV = 0.40 f 0.02 g/lOO g body dominantly expressed and that homozygous mutants weight) and heart (WTV= 0.39 f 0.01 g/100 gbody have the samephenotype as heterozygotes (FAVOR weight) also indicated no difference between wild 1984). The time of death of homozygous mutant types and mutants. Furthermore, the concentration embryos was determined by examining uterine con- of extractable proteins given in g/lOO g wet weight in tents and corpora lutea at 14 days post conceptionem. liver (WTV = 17.1 f 0.8), lung (WTV = 11 .6 f 0.4), All three mutant lines revealed no significant differ- kidney (WTV = 15.3 f 0.5), spleen (WTV = 13.1 f ences in the numberof corpora lutea, preimplantation 0.5), heart (WTV = 10.9 f 0.4), muscle (WTV = 7.8 loss or late death implants between control and inter- f 0.3) and brain (WTV = 6.9f 0.3) were not signifi- crossed dams (Table 2). In contrast, the number of cantly altered in heterozygotes when compared to the live implants was significantly reduced by approxi- wild type. These findings suggest thatneither the mately 25% and the numberof early postimplantation mutations in the heterozygous state per se nor the deaths significantly increased in dams of intercrosses. resulting 42-50% LDH-A deficiency affect physiolog- The mean absolute increasein early death implants is ical functions. Inturn, the results obtained in the approximately as high as the mean absolute decrease hematological tests indicate that thereduction of LDH in live implants, suggesting that lethality of homozy- activity is not anindirect result of an altered redblood gous Ldh-1 mutants generally occurs at an early post- cell population. implantation stage of embryonic development. LDH activities in blood and other tissues: The Physiologicalcharacterization: To determinea levels of LDH activity in blood, erythrocytes, plasma possible effect of LDH-A subunit deficiency on eryth- and several tissuesof wild types and heterozygous rocyte metabolism or conversely to exclude the pos- mutants are shown in Table 3. A comparable LDH sibility thatthe reduced LDH activity in blood is reduction of approximately 42 or 50%as observed in modulated by an altered erythrocyte population, rou- blood and erythrocytes was also found in liver, spleen tine hematological tests were performed. In hetero- and muscle of heterozygous animals. On the contrary, zygous mutants no deviations from the wild-type val- brain, heart and kidney exhibited smaller reductions ues were observed forthe hematocrit (wild-type value in LDH activity ranging from 15 to 30% depending [WTV] = 47.5 k 0.8%),hemoglobin (WTV = 15.2 f on tissue and mutant line, whereas the values of lung 416 S. Merkle et al.

TABLE 2 In utero lethality of homozygotes for three LDH-A deficiency mutations in mice

Dead implants Cross Ldh-1 No. of Corpora Preimlantation Live genotype" females lutea females genotype" LSSb Early Late implants LDH-AZN +/+ x +/+ 11 9.0 f 0.3 0.4 f 0.3 0.6 f 0.2 0.2 f 0.1 7.8 f 0.5 +/- x +/- 11 8.8 f 0.4 0.1 f 0.1 2.9 f 0.4* 0.2 f 0.1 5.6 f 0.3* LDH-A3N +/+ x +/+ 10 11.1 f 0.6 1.1 f 0.3 0.9 f 0.4 0 9.1 f 0.7 +/- x +/- IO 10.7 f 0.8 0.9 f 0.4 3.0 f 0.6* 0 6.8 f 0.5* LDH-A4N +/+ x +/+ 8 9.1 f 0.4 0.6 0.1f 0.4 f 0.1 0.3 f 0.2 8.1 f 0.6 +/- x +/- 8 9.6 f 0.5 0.3 f 0.2 3.4 f 0.4* 0 5.9 f 0.4* Data are given per female as mean f SEM. Significant differences (P I 0.05) between females originating from control crosses and intercrosses are marked by *. a Same symbols are used as in Table 1. For each mutant line, control animals and heterozygous mutants originated from thesame litter. Preimplantation loss was determined using the differencebetween number of corpora lutea and number of total implants.

TABLE 3 subunit occurs show the largest decrease of activity, LDH activity in blood, erythrocytes, plasma and several tissues whereas the predominanceof the B subunit correlates from wild types and heterozygotes for LDH-A deficiency with a minor deficiency in the respective tissue. More- mutations in mice over, a striking consistency was found between het- erozygous animals of the ENU-induced LDH-A3N Ldh-1 genotype' and the radiation-induced LDH-A4N mutants. Mice LDH-APN LDH-A3N LDH-A4N of both mutant lines exhibited essentially the same Tissue +/+ +/- +/- +I- reduction of LDH activity in all tissues studied. The Blood 100.0 f 3.0 58.2f 1.9 50.5f 1.2 49.5 f 2.0 approximate 50% LDH reduction in red blood cells, (226) liver, spleen and muscle corroborates theexclusive or Erythrocyte 100.0 -+ 4.6 57.6 f 2.549.6 f 2.1 49.0 f 2.4 (206) nearly exclusive expression of the LDH-A isozyme in Plasma 100.0 f 9.5 64.4f 8.2 56.8f 6.8 57.8 -+ 9.7 these tissues and suggests that the underlying muta- (0.82) tions generated null alleles at the Ldh-1 locus in both Liver 100.0 f 5.3 58.0f 4.1 52.0f 3.8 49.8f 2.4 mutant lines. However, the occurence of a null allele (1451) Lung 1OO.Of 5.9 67.4f 3.2 58.7 k 3.5 56.3 f 2.9 can beexcluded for the LDH-A2N mutant which (642) expressed approximately 58% activity in tissues con- Kidney 1OO.Of 7.6 82.9f 3.6 73.3-+ 3.5 74.7f 2.4 taining nearly exclusively the A subunit, such as blood, (1 683) liver and spleen. Spleen 100.0 f 2.9 57.0f 3.1 51.9f 3.652.1 f 2.7 Physicochemical and kinetical propertiesof LDH: (789) Heart 100.0 f 4.6 84.6f 3.3 79.4f 1.777.7 f 2.2 The physicochemical and kinetic characteristics of (2228) erythrocyte LDH of A-3N and A-4N mutants includ- Muscle 100.0 f 3.6 61.5f 3.1 57.6f 3.0 58.0f 2.5 ing K, for pyruvate (WTV = 0.200 & 0.002 mM), K, (6796) for NADH (WTV = 36.6 & 2.0 p~),inhibition by Brain 100.0 f 4.9 78.8 f 4.8 72.0f 2.2 71.1 f 2.7 (1353) highconcentrations of pyruvate (8.5 mM: WTV = 74.3 k 2.0%; 20 mM: WTV = 52.0 k 1.2%),substrate Data are expressed as percentageof LDH wild-type activity and given as mean -+ SEM of 10 animals. In parenthesis the meanspecific specifity (glyoxalate/pyruvate: WTV = 3.4 & 0.2%), activity is given as units/g Hb in blood and erythrocytes, units/ml noncompetitive inhibition by oxalate (WTV = 92.5 k in plasma and in units/g protein in other tissues. No significant 0.7%), heat stability at 60.0" and 62.0", pH depend- differences due to genotype were found in the amount of protein extracted per gram tissue. ence, electrophoreticmobility and isoelectric focusing a Same symbols are used as in Table 1. pattern(Figure 1) werenot significantly different from those of the wild type supporting the hypothesis and plasma wereintermediate between both ex- that these mutants are true nulls. In contrast the A- tremes. The differential tissue-specific decreaseof 2N mutant exhibits slight but significant differences LDH activity in themutants thus reflects the well in some of the enzyme properties studied,ie., isoelec- known relative expression of the two LDH isozymes tric focusing pattern (Figure l), heat stability (Figure in murine tissues (MARKERTand URSPRUNC1962). 2), pH dependence (Figure 3) and inhibition by high Organs or cells in which mainly or exclusively the A concentrations of pyruvate (8.5 mM: mutant value LDH-A Subunit Deficiency in Mice 417

is due to diminished specific enzyme activity of mutant LDH molecules.

DISCUSSION Site and nature of the mutations: The detection of a mutant organism exhibiting reduced activity of an enzyme which occurs in more than oneisozymic form always implicates the question as to which isozyme is affected by the mutation. In the case of hereditary LDH deficiency in the murine blood, however, the answer is evident a priori. Because of the predominant occurrence of the LDH-A isozyme in mouse erythro- cytes (ENGEL,KREUTZ and WOLF 1972), a detectable LDH deficiency in blood of this species can be attrib- uted to a deficiency of A subunits caused by a muta- tion affecting the Ldh-l structural locus. In the case of the two ENU-induced mouse mutants 1,DH-A2N and LDH-ASNthis has been confirmed by linkage analysis (PRETSCH1989); the genetic confirmationwas notperformed in theradiation-induced LDH-A4N mutantline but the extensive biochemicalanalyses presented here confirm this hypothesis. Regarding the mutantswith approximately 50% of normal LDH activity, LDH-ASN and I,DH-A4N, the study of physicochemical, kinetic and immunological properties as well as the results of the enzymeactivity measurelrlerlts in a variety of tissues suggest mulatiom generating nullalleles with neither detectable enzyme FIGUREI .-Isoelectric focusing pattern of erythrocyte LDH of activity nor immunologically cross-reacting material. wild types and heterozygotes for threeLdh-1 mutants. Lane I, Ldh- However, since null alleles may result from a broad 1"jLdh-1': lane 2. Ldh-I"jLdh-I"'"'~'"; lane 3. Ldh-I"jLdh-l""'~'": spectrum of DNA alterations including nonsensemu- Ime 4. Ldh-1"lLdh-1"'"~''". tations, frame shifts or deletions, these findings may only give limited insight into the nature of the muta- [MV]= 83.7 f 0.9%; 20 mM: MV = 64.0 & 1.2%). tions. Yet, some deductions as to the latter might be Immunological properties: To confirmthe hy- made from the origin of the mutations. Determination pothesis that LDH-A3N and LDH-A4N are null mu- of the exact natureof ENU-induced mutations by tations and that LDH-A2Nis different from the other DNA-sequence analysis showed that the predominant two, Western blotanalysis was conducted using spleen mutationswere base pairexchanges (GOSEN et al. tissue since it exhibits the same LDH isozyme distri- 1989; PASTINKet al. 1989; SIMONSet al. 1991). In bution patternas in blood and erythrocytes (MARKERT contrast, the mutation spectrum following exposure and URSPRUNG 1962).As seen in Figure 4, Western to X-rays is more complex. As summarized by GRO- blots reveal similar molecular sizes of the detectable SOVSKY et al. (1988) several reports estimate the per- centageof alterations detectable by Southern blot LDH enzymes and no additional bandas compared to analysis (>50 bp)between 16 and80% in ionizing the wild type. Further, the quantitativeanalysis (Table radiation-inducedmutations of mammalian cells. 4) clearly demonstrates that the amount of detectable Among the large number of radiation-induced muta- LDH is similar in wild types and LDH-A2N mutants, tions alterations ofwhich are not detectableby South- whereas it is about half in LDH-A3N and LDH-A4N ern blot analysis (<50 bp), the majority (about 2/3) animals. The comparison between LDH activity and represents base substitutions. the amount of LDI I-cross-reacting material in LDH- Physiological effects of LDH-A subunit defi- ASN and I.DH-A4Nindividuals suggests that the ciency: Even if the defined nature of the mutations decrease of LDH activity in these two mutants repre- encountered in this study could not be elucidated in sents a reduction in immunologically detectable pro- detail there seemsto be yet sufficient evidence that at tein. Moreover, it indicates that the decrcasc of LDH least two of the three LDH mupants are the rcsult of activity in LDH-A'LN mutants is not a resultof a mutations affecting exclusively the Ldh-I structural reduced concentration of LDH molecules, but rather gene. Consequently, the resultant physiological phe- 418 S. Merkle et al.

+

60 -

40 -

20 - 20 -

0 I 1 5 10 1s 20 5 10 1s 20

Time (rnin) FIGURE2.-Percent residual activity of erythrocyte LDH of wild types (open circles) and heterozygous Ldh-ZamZN"mutants (closed circles) after incubation at 60" (left) and 62" (right). Values are given as means of 6 animals (double determination). Bars represent flSEM where visible, or are smaller than the plotted symbol.

0' 1 I 8 6.5 7 7.5 8 0.5 9 9.5 10

FIGURE 3.-Percent of maximal activity of erythrocyte LDH of wild types (open circles) and heterozygous Ldh-Z"'"2N" mutants (closed circles) at different pH values. Values are given as means of six animals (double determination). Bars represent fl SEM where visible, or are smaller than the plotted symbol. notype of the mice carrying the respective mutations metabolic symptoms (STANBURY,WYNGAARDEN and should be attributable to aclear cause-effect relation- FREDRICKSON1972). Notsurprisingly, the 42-50% ship between enzyme deficiency and metabolic func- reduction of LDH-A activity in heterozygous Ldh-1 tion. mutants given in thispaper produce no obvious harm The presence or severity of a metabolic dysfunction to the carriers. In contrast, the expected total loss of depends on the degree of the enzyme deficiency.The LDH-A activity in homozygous LDH-A3N or LDH- study of enzyme deficiency and hereditary disease in A4N animals, as well as the expected severe LDH-A humans presented evidence that at least for enzymes deficiency in LDH-AQN homozygotes was shown to other than those which are rate-limiting, more than have lethal consequences for these animals as early 50% reduction ofactivity is necessary to produce postimplantation embryos. LDH-A Subunit Deficiencv in X4ice 419

a b oocyte-codedfrom to embryo-codedenzyme in the

. . ?_" developingembryo. kD2 The roles of 1,DH within the mammalian metabo- lism are well understood (I'F~SEL197.5; EVERSEand KAPLAN 1975). Oneof the most important is the regeneration of NAD+ required for glycolysis to con- tinue under anaerobic conditions. Since under these 66- conditions the glycolytic pathway is the ma-jor source of cellular ATP and moreover the LDH step, with few exceptions, may not be compensated, deleterious 45- consequences due to I.DH deficiency areexpected under environmental orfunctional anaerobiosis. Although there havebeen no measurements of 36 - oxygen availability in the earlypostimplantation mouseembryos it may beassumed that until the 29- formation of the chorioallantoic placenta, the embry- 24 - onic tissue is almost certainly developing under anaer- 20 - obic conditions (ELLINGTON1987). Accordingly, pre- 14- viotls metabolic studies suggested that the early post- 1 234 432 1 implantationembrvomouse unlike the FIGURE4.--\Yestern blot analysis of splenic LDH of wild types preimplantation lnouSe embryo ciepends upon glvcol- andheterozygotes for three Ldh-1 mutants. SDS-PACE of spleen ysis for survival (CLOCCH ;id \~'Hl'ITlNGHAM 1983; proteins. a) Staining with Coomassiebrilliant blue. Irlolecular mass C,ARDNERand 1,~p-s~1988). 'I'his suggestion $vas sup markers are: bovine albumin (66 kD);egg albumin (4.5 kD); glyc- ported by the finding that mice homozygous for both er;\ldehyde-3-pho~phatedehydrogenase (36 kD): carbonicanhy- (29 kn); trypsinogen (24 trypsin inhibitor (20 kl)); Q- Tpi-I and Gpi-Is null alleles dieat an earlypostim- lactalbumin (14 kD).b) Analysis with LDH-specific antibodies. file plantation stage of embryonic development (h1ERKI.E 1.1)Il-specific band appears at 36 kD (molecular mass ofI-DH and PRETSCH 1989; M'FST tt al. 1990). subunik). Inne I, Ldh:I"/Ldh-l";lane 2, Ldh-I"/Ldh-l"~""";lane In comparison with TPI andGPI, which only occur 3. lane 4. Ldh-l"/Ldh-l""'"": Ldh-I"/Ldh-l"~"~"". in one isozvmic form. the situation for LDH is more The prediction whether and at which stage of on- complexbecause ofthe presence of two isozymic togenic development a mutational event generatinga forms in most tissues. Because the deficiency of the severe deficiency or total absence of a given enzymatic one enzyme due to a mutation could be easily com- gene product may have deleterious consequences for pensated by the persistent presence of the second. the metabolism and may result in lethality of the or&?- tissue-specific and/orthe developmentalpattern of nism, is predicted upon the consideration of several the isozymes must be taken into consideration. More- points: (I) the role of the enzyme and of the corre- over. one might argue that LBH appears to play an sponding metabolic pathway within the total metabo- important role in the early preimplantation embryo. lism of an organism, (2) the possibility of alternative During the two to eight cell stage when the embryo pathways or of bypasses of the respective enzymatic passes throughthe lactate-rich environment of the step in theaffected pathway, (3) thepresence of Fallopian tube, pyruvate appearsto provide the ma-jor isozymes as well as their tissue-specific and develop- energy source for the mouse and other mammalian mentalpatterns and (4) theontogenetic transition embryos (BRINSTER1965, 1970; LEF-SE and BARTON

TABLE 4 Intensity of LDH-specific bands after Western blot analysis and the specificLDH activity in the spleenof heterozygotes for three LDH-A deficiency mutations in mice

.Acrivirv +/+ I.I)H-ABS +/- I.Dfl-.i\fS +/- I.DlI-A4S +/- Intensity of LDH-specific band (% of wild type) 100.0 It 13.3 100.6 f 5.9 52.9 * 3.7* 59.4 f 7.0' I.I)H activity (V/g protein) 840.4 f 60.6 485.8 f 58.08 467.3 -c 35.38 486.6 -I 95.88 Specific LDH activity (% of wild type)A 100.0 -I 10.5 5725 * 3. I * 105.9 * 8.6 97.5 f 12.1

Data are given ;IS mean f SEM of four animals. Significant differences between wild tvpes and mutants are markedhy *. Same symtmls are used as in Table 1. * Specific I.DH activity was calculated by relating the LDlI activity of spleen given in units/g protein to the intensity of I.DI1-specific band. 420 S. Merkle et al.

1984). Oxidation of exogenous lactate by embryonic for the impact of a mutation on physiological func- LDH could be an important source of pyruvate. An tions. effect of LDH-A deficiency on ontogenetic develop- ment of the preimplantation mouse, however, is not We would like to express our appreciation to U. H. EHLINGfor discussions and helpful criticism of the manuscript. The competent to be expected since LDH activity during this phase technical assistance of M. ELLENDORFF,S. WOLF and E. BURKLE is is contributedfrom oocyte-coded LDH-B subunits gratefully acknowledged. We also thank G. GODDENGfor preparing (for areview, see BICCERSand STERN1973; BRINSTER the photographs. This research was supported in part by Contract 1973, 1979). This predominance of the B subunit is B16-E-156-D from the Commission of the European Communities. lost during implantation when an increase in the syn- LITERATURE CITED thesis of presumably embryo-coded A subunits results in a shift in the isozyme pattern such that 90% of the AUERBACH,S., and R. L. BRINSTER,1967 Lactate dehydrogenase LDH activity is contributed by the A subunit (AUER- isozymes in the early mouse embryo. Exp. Cell Res. 46 89-92. BEUTLER,E., C. K. MATHAI and J. E. SMITH,1968 Biochemical BACH and BRINSTER1967; RAPOLAand KOSKIMIES variants of glucose-6-phosphate dehydrogenase giving rise to 1967; BRINSTER1970; MONKand ANSELL1976). As congenital nonspherocytic hemolytic disease. Blood 31: 131- development proceeds, the contributionof B subunits 150. tothe pattern increases (MARKERTand URSPRUNC BIGGERS,J. D., and S. STERN,1973 Metabolism of the preimplan- 1962; ENCELand PETZOLD1973; MONKand ANSELL tation mammalian embryo. Adv. Reprod. Physiol. 6 1-60. BRINSTER,R. 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