Ribonucleic Acid and Protein Metabolism of t12/t1* Embryos and T/ti* Spermatozoa R. P. Erickson, C. J. Betlach and C. J. Epstein Department of Pediatrics, University of California, San Francisco, California 94143, U.S.A.

RNA precursor uptake and incorporation, amino acid uptake and incorporation, and the characterization of newly synthesized RNA and protein in pools of normal morulae and pools containing one-third t l z / t l * morulae were compared. Maturing spermatoza of +/+ and T/ f lZ

animals were analyzed for RNA and protein content, and the RNA characterized. No differences in these parameters could be ascribed to the f 1 2 gene in homozygous embryos or haploid sperm.

Introduction

Many of the t-alleles of mice are embryonic lethals when homozygous.1 Most are abnormal in their genetic transmission in males and cause a marked distortion of the Mendelian 1 :I allelic segretation ration.* One of the most studied of these alleles is tlz. It is also one of the earliest acting embryonic lethal alleles known, develop- ment ceasing at the late morulae stage.2 Early morphological and histochemical studies2-3 sug- gested that abnormalities of ribosomal RNA synthesis might cause this developmental arrest; and this seemed to be corroborated by a report of decreased binding sites for ribosomal RNA with hemizygous t 1 2 DNA.4 However, more recent electron microscopical,^ autoradio- graphic,6 and biochemical studies7 have not disclosed an abnormality of RNA synthesis or processing in tlZ/t12 embryos.

In Drosophila spermatozoa, the lack of a nucleolar organizer has been related to segre- gation distortion,* and there have been many similarities between the t-alleles and the major segregation distortion locus, SD, in Drosophila.9 Since a unitary hypothesis of gene action would indicate that the effects of the t12 allele in homo- zygous embryos and hemizygous sperm should be related, we have studied RNA and protein metabolism of pools of embryos containing t 1 2 / t 1 2 embryos, and the spermatozoa of T/t12 males.

Methods

Mice The Tlt12 stock was obtained from Dr S. Gluecksohn-Waelsch of Albert Einstein College

Diflereatiation 2, 1974

of Medicine, Bronx, N.Y. and maintained by brother-sister matings. The mice were main- tained on a 12 h dark, 12 h light cycle and shavings were used as bedding,

Embryos The Tit 12 stock was crossed to Swiss-Webster random bred mice (local dealers) and the T / t (short-tailed) and t 1 2 / + (normal-tailed) off- spring obtained. The young female t12/+ mice were hormonally treated to induce super- ovulationl*,ll and then mated to t ’* /+ or T/+ males, to produce pools containing t12/t12 homozygotes or ‘normals’ (t12/+, +/+, Ti+, T/t12), which are genetically similar by strain background (F2s of the T/tlz by Swiss F1). Day 2 embryos (morulae) were used for all the bio- chemical studies.

Embryo RNA studies Quantitation of the rate of uptake of labelled precursors into embryos was performed during in vitro incubation as previously described.12 The extent of incorporation of the precursors into RNA was determined by a modification12 of the base hydrolysis microtechnique of Kennel.13 Polyacrylamide gel electrophoresis of labelled RNA was performed on 2.5% acrylamide- bisacrylamide gels run for 2 h at SmA/tube. Frozen gels were sliced into 1 mm fractions, digested in 1 ml NCS, and counted.14

Embryo protein sfudies Quantification of the rate of uptake of 3H-leucine was performed during in vitra incubation as previously described.15 The extent of incorpora-

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R. P. Erickson, C. J. Betlach and C. J. Epstein

tion of 3H-leucine into protein was determined by a modification15 of the method of Kennel.13 Polyacrylamide gel electrophoresis of 3H-tyrosine pulse-labelled proteins was performed by heating the washed embryos in 0.0625 M Tris HC1 (pH 6.8), 2% sodium dodecyl sulphate, 10% glycerol, 5 % 2-mercaptoethanol and 0.001 % bromphenol blue for 1 . 5 min at 100°C. The proteins were then electrophoresed according to Laernmli16 on 8 % gels with 3 7; stacking gels for 3 h at l.SmA/tube.

ATP assays The embryonic content of ATP was determined by the luciferin-luciferase assay as previously described. 17

Spermafozoal methods Maturing Tit12 spermatozoa were obtained from the epididymides and vasa deferentes by the slicing, screening, and washing technique pre- viously described.18 Spermatozoa1 RNA was determined by the orcinol method and extracted for polyacrylamide disc gel electrophoresis by the methods of Betlach and Erickson.19 Protein determinations were by the method of Lowry, et a1.20

Results

The segregation ratio of t12 to T was observed among 288 offspring of Swiss females and T/t12 males, and a 2:1, t12:Tratio was found (Table l), which is somewhat lower than the ratio of 3: l reported by Smith.2 This presumably represents a further change in the strain-average segregation ratio: there is marked heterogeneity between males for segregation ratiosl, while there is no effect of the female genotype,z’ so the lower

ratio found is unlikely to be due to the Swiss Webster females. For comparison, the normal 1 :1 , t12-T ratio in the offspring of T/tl2 females mated to Swiss males, and the normal sex ratio for both crosses, are presented. This segregation ratio leads one to expect that one-third of the embryos obtained in crosses of t12/+ males are rlZlt12. Since it is possible that the superovulation procedures could alter the ratio, embryos were harvested on day 3 and studied under the dis- secting microscope. Table 1 shows that the t l 2 / +

infer se mating resulted i n 25.5% arrested morula, 49 % blastocysts, and 25 ”/, degenerated embryos, while there were no arrested morula, 74.5 74 blastocysts and 25.5 % degenerated embryos in the control mating. Correcting for the degenerated embryos (which are always culled out for the biochemical work), 34 :’ of the non-degenerate embryos from the t12/+ intfr se mating are arrested morulae as expected of tlzjt12 embryos. The arrested morula were cultered in vitro for 2 days and only one blastu- lated, all others degenerating.

The uptake and incorporation of adenine and uridine showed no significant differences between pools of embryos with one-third t12/t12 and no t12/t12 embryos (Table 2). The experiments vary somewhat on different days so that the standard errors for the replicates on uridine uptake are high. This tends to hide any tl2/t12 abnormality, but single uptake and incorporation deter- minations with adenine, and replicates on uridiiie incorporation, did not suggest any major differences. More significantly, the gels of the purified RNA from embryos pulsed for 2 h with 3H-uridine showed very similar profiles for 28s (slice about No. 14), 18s (slice a b w t NO. 24) and transfer RNA (slice about No. 53), Fig. 1.

The day 2 embryo pools, with and without tl~/t12 embryos, also showed no differences in 3H-leucine uptake or incorporation (Table 3).

Table 1 T/t12 Segregation distortion and pre-implantation lethality*

Segregation t12 T (1) T / f ‘ 2 x s w (2) s w x T/t’*

214 : 220 192 : 96

S Y 224 : 210 127 : 161

Lethality Arrested morula Blastocysts Degenerated (1) fl’/+ X t”/+ 29 = 25.5% (34”/,)** 56 = 49% 29 = 25.5%

(2) t12/ t x Ti+ 0 47 = 74.5% 16 = 25.5% expect 33%

* Female listed first in crosses. ** % of arrested morula and blastocysts in parentheses.

204 Differentiation 2, 1974

t12-Related RNA and Protein Metabolism

Table 2 Uptake and incorporation of RNA precursors by normal and t12/t12 containing pools of embryos

cpmlembryolh Normal t’Z/t’Z pool

Precursor Uptake Incorporation Uptake Incorporation Adenine 10,110 21 4 11,350 294 Uridine 211 & 27” (5) 12.7, 27.6 157 f 47 (5) 16.8, 5.0

* Mean f standard error (number of experiments).

In addition, acrylamide gel electrophoretograms of 3H-tyrosine pulse-labelled proteins from the two p3ols showed almost perfect congruence (Fig. 2).

The levels of ATP measured on ‘normal’ or tl2/t 12-containing pools of embryos from the t NO crosses, showed quite a lot of scatter. They were 0.34 + 0.17 pmol/ernbryo (mean and standard error for 8 pools) in the ‘normal’ and 0.73 + 0.26 (mean and standard error for 6 pools) in the tl2/t12-containing embryos.

The overall composition of T/f 12 spermatozoa was not detectably different from that of +/+ spermatozoa (Table 4). The DNA determinations (same in both cases) serve as a control for any possible differences in preparation, washing, or lyophilization.

Thus, the similar protein and RNA contents are valid. The RNA from the two kinds of spermatozoa was further compared by SDS-gel electrophoresis of extracted RNA. Again, there was no qualitatively detectable difference in the RNA from T/f12 compared with +/+ sperma- tozoa. A characteristic 11s degradation product (due to the action of a spermatozoa1 ribonuclease)

was present in both cases, and mixing showed no electrophoretic differences (Fig. 3).

Discussion

Smith’s morphological, histological and histo- chemical methods2 revealed that t Iz/f 12 embryos effectively stop development as 30-cell mxulae about 80 h after fertilization. The concentration of cytoplasmic (presumably ribosomal) RNA was less in the mutants than in normal litter- mates.2 The size of mutant cell nuclei increased and the nucleoli were abnormal, as they did not assume an irregular shape, lose their vacuoliza- tion, and occupy a large part of the nucleus. It was soon shown that the same defects occurred with in vitro cultivated embryos, and that actinomycin D treatment cnused quite different defects.22.23 Chimera formation between normal and t l z / t 12

mid-cleavage embryos demonstrated the cellular autonomy of the t 1 2 / t ” defect.24 The electron microscopical studies of Calarco and Brown25 confirmed Smith’s findings. It was apparent that these changes may have been secondary, although

I

4-5s

--- I ’ t ’ 2 / P pool ‘ I

- NORMAL

0 10 20 30 40 50 60 70 80

GEL SLICE No.

Fig. 1 Graphs of the polyacrylamide gel electrophoretograms for purified RNA following a 2 h 3H-uridine pulse from normal (dotted line) andl’2/t12-containing pools (continuous line) of embryos.

Diferentiation 2, 1974 205

R. P. Erickson, C. J. Betlach and C. J. Epstein

Table 3 Uptake and incorporation of 3H-leucine by normal and P2/P2-containing pools of embryos

cpm /embryo//, Normal t'2/t'2 pool

Uptake Incorporation Uptake Incorporation 435 f 17* (4) 87.7 f 10.4 (4) 381 f 47 (4) 102.2 22.2(4)

* Mean & standard error (number of experiments).

the intranuclear bodies were similar to those observed in the anucleolate nuclei of mutant Xenopuszb and Zea muys.27

The later morphological findings,s which did not support the conclusion that t 1 2 / t 1 2 embryos showed a defect in RNA synthesis, were buttressed by autoradiographic studies, which found no difference in 3H-uridine uptake and incorporation between t 1 2 / t 12 and normal

embryos.6 Better quantitative data were ob- tained by studies on extracted RNA from pools of +/+ or + /+ and t l Z / t * 2 radio-labelled embryos.7 No qualitative differences in RNA (separated by density gradient centrifugation) were found between the two sources of RNA. We have extended these studies by using several RNA precursors, more sensitive methods of characterizing RNA, studying protein synthesis,

GEL SLICE NO.

206

Fig. 2 Graphs of the polyacrylamide gel electrophoretograms for proteins following a 2 h 3H- tyrosine pulse from normal (dotted line) and t12/f12-containing pools (continuous line) of embryos.

Differentiation 2, 1914

and checking some of these parameters with spermatozoa as well. We have performed all the embryo experiments with 8-16 cell stage embryos (day 2) as this is shortly before the t12/t 12 develop- ment arrest of the latemorulaestage(day 3), and is the time at which any casual aberrations of metabolism should be occurring.

Table 4 Sperm composition related to T-alleles (determined on lyophilized sperm).

Swiss T/tl* Protein (Lowry) 58% 57%

DNA (diphenylamine) 16% 16% R N A( o rci nol) 1% 1 %

The uptake of uridine by day 2 embryos is very low, while a three-fold increase occurs on day 3.12 Thus, moderate differences in the time of embryo harvest and incubations can result in large fluctuations in the absolute values obtained. The many replicates performed in these experiments had a large standard error (Table 2), reflecting this problem; but no statistically significant differences were found between the t12/tl2- containing and normal pools. There was also a moderate scatter in the incorporation values, but the pairs of values overlap each other. The adenine uptake curve shows a similar increase from day 2 to 317 (but at much higher levels). Single values showed no difference between the tlz/t 12-containing and normal embryo pools.

Studies on RNA synthesis in the early mouse embryos have demonstrated transfer RNA and ribosomal RNA synthesis as early as the 4-cell stage of cleavage.28-31 This was also found clearly in the polyacrylamide gel electrophoreto- grams of newly synthesized RNA from 8-16 cell embryos, where 28S, 18s and 4s components can easily be observed (Fig. 1 j. Again, this very sensitive method disclosed no significant differ- ences between t lz/f 12-containing and normal pools of embryos; the slight increase in the 28s and 18s peaks for the tltlt12-containing pool would represent about a 25% increase in the tlzlt12 embryos, if limited to them. The change is at the limit of replicate runs for one sample and suggests that there certainly is no deficiency of synthesis of ribosomal RNA in t12/f12 embryos.

Leucine uptake is low in day 2 embryos and increases about three-fold by day 3.15 Hence, there is also a large standard error for multiple- determinations of leucine-uptake and incorpora- tion on day 2, but no statistically significant

Diferentiation 2, 1974

tl2-Related RNA and Protein Metabolism

differences were found between the mutant and normal pools of embryos (Table 3). As with RNA, polyacrylamide gel electrophoresis of the newly synthesized macromolecules provides a sensitive indicator for altered protein metabolism. Again, no differences were found in eight peaks representing multiple protein (Fig, 2).

a b C

about species

d Fig. 3 Polyacrylamide gel electrophoretograms comparing RNA extracted from normal (Swiss) and T/f12 spermatozoa; (a) Swiss sperm RNA; (b) Swiss sperm RNA -i T/t12 sperm RNA; (c) TIP2 sperm RNA; and (d) liver RNA for comparison. The arrow indicates the position of the 11s band.

Energy metabolism has been characterized by studies on ATP and ADP levels in pre- implantation embryos of the rabbit32 and mouse.33 The latter authors have claimed that there are lower levels of ATP in tlZ/t12 than in normal embryos prior to the stage when most t12/t1* embryos die.34 Although we have found considerable scatter in the determinations, we find no significant differences between t 12/t12- containing and normal pools of embryos,

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R. P. Erickson, c. J. Betlach and C. J. Epstein

although the trend is towards higher rather than lower levels in the t 1 2 / t 1 2 embryos.

We found no difference in the amount of pro- tein or RNA in T/t'Z compared with +/+ spermatozoa. Although previous results were considered to indicate only mitochondria1 RNA metabolism in ejaculated sperrnatozoa,35 we have found evidence for 28s and 18s in maturing mouse spermatozoa (Betlach & Erickson, in preparation), and others claim that these com- components are metabolically active in maturing rat and hamster sperm.36 We have found that a spermatozoa1 ribonuclease rapidly degrades these components to a 11s component during phenol- SDS extraction19 (also Hickum & Erickson, in preparation). The failure to find any differences between T / t 1 2 and +/+ RNA, even when mixed and co-electrophoresed (no band splitting, Fig. 3b), suggests that the KNA is qualitatively, as well as quantitatively identical in the two situations.

It thus seems that there is probably no primary

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defect in major components of RNA or protein metabolism in tl2/t12 embryos, or in RNA com- position of T/t12 spermatozoa. These studies certainly would not detect any changes in messenger RNAs or in individual tRNAs but should, along with the work of Hillman's group,5-7 lay to rest the notion of a primary defect in ribosomal RNA metabolism. We predicted sometime ago1 that cell surface changes might well be the common denominator of t-allele effects and the recent report of a T-antigen37 would seem to support this view.

This work was supported by grants from the National Institutes of Health, Bethesda, Md. (HD- 05259 and HD-03132). R. P. E. is the recipient of a Research Career Development Award from the National Institutes of Child Health and Human Development.

Received: January, 1974

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