DNA synthesizing activity in normal human sperm

Experimental

Cell Research 106 (1977) 47-54

DNA SYNTHESIZING

ACTIVITY

HUMAN

SPERM

Location and Characterization S. S. WITKIN

TN NORMAL

of the Endogetzous Reaction

and A. BENDICH

Laborutory of Cell Biochemistry, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA

SUMMARY Chromatin-associated complexes have been purified from membrane-free nuclei of normal human sperm heads. They band at a sucrose buoyant density of 1.1.5-l. 19g/ml and contain macromolecular components which catalyze the endogenous synthesis of DNA in a reaction partially sensitive to ribonuclease pretreatment. The sedimentation rate and buoyant density of the purified reaction product, still attached to its endogenous template, demonstrate that the [3H]DNA is associated with a high molecular weight RNA, which may be serving as both template and primer.

DNA polymerase activity in the mature male gamete has received scant attention, owing perhaps to the assumption that the sperm nucleus was inert. Although four distinct DNA polymerases have been partially purified from testes (mouse) [l], and three from early stage embryos and trophoblasts (mouse) [2], only the polymerase present within tail mitochondria has been identified in mature mammalian sperm (bovine) [3]. The cytological demonstration of DNA polymerase activity in epididymal sperm nuclei (mouse) has recently been reported [4]. Indirect evidence does exist for the occurrence of DNA polymerase activity in sperm nuclei. Several workers have utilized radioactive precursors to demonstrate a low level of isotope incorporation into DNA of male pachytene stage meiotic cells of mouse 1.5,61, lily [7], and man [8]. In Triturus, the 4-771816

exceptionally long spermatogonial S phase preceding mitosis has been ascribed to a gross reduction in the number of initiation points for replication and not to a decreased rate of polymerization [9]. Even at the later mid-spermatid stage, where DNA synthesis normally does not occur, re-initiation of DNA polymerase activity was demonstrable following treatment of mice with ethyl methanesulfonate [lo]. Brief, albeit unsubstantiated, reports on [Vjglycine incorporation into the DNA of in vitro incubated ejaculated bovine sperm have also appeared [ 111.The chromatin released from chemically decondensed ejaculated sperm has been shown capable of serving as a template for exogenously added bacterial DNA polymerase [ 121. We recently presented data suggesting the occurrence of DNA-synthesizing complexes in human cell-free seminal fluid [l3] Exp Cell Krs 106 (!F77)

48

Witkin and Bendich

and in association with human sperm nuclei [13-151. Suspension of a purified population of sperm heads in 0.02 M dithiothreitol resulted

in the swelling

of the nuclei

and

release of ribonuclease-sensitive DNAsynthesizing complexes [13, 141. The buoyant density of the liberated complex depended upon the extent of treatment of the sperm heads with dithiothreitol . Incubation with dithiothreitol for 20 min resulted in DNA polymerizing activity which banded at a sucrose density of 1.21-1.25 g/ml [13], while treatment for 180 min led to banding at a density of 1.15-1,19 g/ml [14]. Further incubation resulted in no additional change in density; no activity was observed in nontreated sperm heads [ 131. Dithiothreitol treatment cleaves disulfide bonds between cross-linked adjacent sperm protamines [ 16, 171, causing decondensation of the nuclear chromatin and nuclear swelling [ 151. This treatment initiated the release of DNAsynthesizing complexes, some of which were perhaps associated with chromatin fragments. The progressive lowering of buoyant density which attended the increased dithiothreitol treatment suggested that release of the complex was accomparried by its detachment from dense chromatin components. Evidence is provided in this communication demonstrating that the DNA synthesizing complex, associated with sperm heads of all individuals examined [14], is situated within the membrane-free nucleus and is associated with a high molecular weight RNA component which may function as both template and primer for the synthesis of DNA.

MATERIALS

AND METHODS

Human semen was obtai@ from medical student donors. Following liquefaction (20 min, 22”C), an equal volume of glycerol was added and the samples stored at -20°C. Exp Cd Res 106 (1977)

All chemicals were of the highest purity available. Unlabeled deoxyribonucleoside triphosphates were from Calbiochem (La Jolla, Calif.). [Methyl-3H]deoxythymidine-5’-triphosphate, 17.5 Ciimmole was from Amersham/Searle (Arlington Heights, Ill.). Trypsin inhibitor (type I-S, chromatogmphically prepared from soybean) and phospholipase C (type 1, from Clostridium wekhii) were purchased from Sigma (St Louis, MO). Worthington Biochemical (Freehold, N.J.) supplied trypsin (3 x crystallized from bovine pancreas, 200 Ulmg), ribonuclease I (from bovine pancreas, electrophoretically pure, 5 139 U/mg) and deoxyribonuclease I (chromatographically prepared from bovine pancreas, 2600 U/mg). The ribonuclease was heated at 100°Cfor 10 min prior to use. The purification of membrane-free sperm heads by treatment with 1.5 % Sarkosyl and sedimentation through a column of 60 % (w/w) sucrose was as previously described [ 13, 141. Sperm heads were swollen by gentle homogenization in 0.05 M Tris-HCl, pH 7.5, plus 0.02 M dithiothreitol and incubated at 0°C for the times indicated in the individual experiments. Dissolution of the chromatin was accomplished by the addition of 100 yglml trypsin followed, after 5 min at 37°C by the sequential addition of 200 pglml soybean trypsin inhibitor, 10 pg/ml deoxyribonuclease and 2 mM Mg2+. After an additional 15 min at 37°C the sample was chilled, and centrifuged at 4°C for 10 min at 10000 g. Centrifugation of the resultant supematant through 15-60% (w/w) sucrose gradients and fraction collection were as reported [13, 141. Endogenous DNA polymerase activity in the fractions was determined by the addition of 25 ~1 spermderived aliquots to the following mixture: 20 nmoles each of dATP, dCTP and dGTP, 1.9 ,umoles MgC12, 0.5 pmoles dithiothreitol, 0.13 @moles Tris-HCl, pH 7.5 and 4 nmoles of dTTP plus 5 &i [3H]dTTP (17.5 c.1 I mmole, 805 cpm/pmole final spec. act.). The final volume was 0.125 ml. Following incubation at 37°C for 15 min, 10 pg yeast RNA plus 2 ml cold 5% (TCA) containing 1% Na pyrophosphate were added, and acid-precipitable radioactivity determined [ 131.Blanks consisting of all reaction components except spermderived fractions were incubated at the same time as were the test samples and the r3H]dTTP incorporated (20-100 cpm) subtracted from the experimental values. Detergent was not needed in the reaction mixture to elicit activity since the sperm had already come into contact with Sarkosyl; complexes obtained from whole sperm without the use of a detergent step showed enhanced activity in the presence of 0.01% Triton X-100 (unpublished). Ribonuclease sensitivity of the endogenous reaction was measured by pooling the active fraction from a sucrose gradient, preparing a 165000~ pellet by centrifugation for 60 min, and resuspending in 0.2 ml 0.05 M Tris-HCl, pH 7.5, 0.02 M dithiothreitol. The sample was divided into two equal parts and incubated at 22°C for 30 min in 7.5 pmoles MgCl,, 10 pmoles NaCl in eithtr. the presence or. absence - -.- -of !jOlg/rpJ pancreattc ribonuclease I. The samples were then chilled to 0°C diluted to a final volume of 0.5 ml with 80 nmoles each dATP, dCTP, dGTP, 40 fig actinomycin D and 100 j&i r3H]dTTP (15.7 Cilmmole) and incubated at 37°C for 10 min. The endogenous product-

Human sperm DNA-synthesizirzg template complex was purified by the addition of 1% sodium dodecyl sulfate, 0.1 M NaCl and an equal volume of phenol previously equilibrated with 0.05 M Tris-HCl, PH 7.5. The actueousphase was laveredonto IO-30% s-&bilking glycerol gradients, subjected to velocity centrifugation in an SW 5OL rotor at 200 000 g for 90 min, fractionated from below and acid-insoluble radioactivity determined on 50 ~1 aliquots. For CspSOl gradient analysis, active fractions from a sucrose gradient were pelleted, resuspended in 0.09 ml 0.05 M Tris-HCl, pH 7.5, 0.02 M dithiothreitol and divided into 0.06 ml and 0.03 ml aliquots. Both samples were incubated at 22°C for 30 min in 7.5 pmoles MgClz, 10 pmoles N&l, 0.02% Triton X-100; 3 pg pancreatic ribonuclease I was also added to the smaller sample. Both samples were then added to DNA-synthesizing reaction mixtures, incubated at 37°C for 10 min, and nucleic acids purified, as described above. Following ethanol nreciuitation. the samples were resuspended in 0.01 MLTrisHCl, pH 7.5, 0.002 M EDTA. The PHlDNA product svnthesized in the absence ofribonucl~se was-divided into two equal oarts. one of which was heated at 100°C for 10 min followed by rapid cooling in ice. All three samples were then brought to 2.0 ml with 0.01 M Tris. 0.002 M EDTA, an equal volume of saturated Cs,SO, solution in the same buffer was added, and the samples centrifuged in an SW56 rotor at 86000 g for 65 h at 15°C. Fractions were collected from below, density determined from refractive indices, and the acid-precipitable radioactivity of each fraction determined.

RESULTS Location of sperm-derived synthesizing complex

DNA

Treatment of human sperm with the anionic detergent, Sarkosyl, results in decapitation [18] plus the complete removal of plasma and acrosomal membranes [ 151. The sperm head morphology is still maintained, however, by a network of cross-linked chromatin [15]. Experiments were performed to ascertain whether enzymatic digestion of the sperm chromatin would lead to an increased yield of DNA-synthesizing complex. A purified suspension of detergent-treated, membrane-free sperm heads was swollen by incubation in 0.02 M dithiothreitol (O°Cfor 90 min), and the preparation was divided into two equal aliquots. One received trypsin (100 pglrnl) and both were incubated at 37°C. After 5 min, soybean trypsin inhibitor

complex

49

(200 pg/ml) plus pancreatic deoxyr nuclease (10 pglml) was added to the trypsin-containing sample and incubation of both samples continued for an additional 15 min. At the end of the incubation: swollen intact sperm heads were still present in the dithiothreitol-treated sample while in the trypsin deoxyribonuclease-treated sample only fragments of the sperm heads were seen. Observation was by phase csntrast microscopy. Following centrifugation of both the treated and untreated sperm head preparations at 10000 g, the supernatants were layered on Linear 15-&I% (w/w) sucrose gradients and centrifuged for I6 h. In the sample treated only with di threitol, endogenous DNA polymerase activity was mainly distributed at two density regions; the majority of the activity banded at a density of 1.25 g/ml whife a lesser amount was located at 1.I7 g/ml (fig. I). The addition of trypsin and deoxyribonuclease to the swollen head preparation resulted in the emergence of the activity at the 1.15 g/ml density region as the predomimant peak (fig. 1). In addition, the total a present in this latter gradient was seven-fold greater than that with the nonenzyme-treated sample. Similar density shifts of DNA-polymerizing fractions from 1.25 to 1.15 g/ml and enhanced total activity were also observed by protongation of the dithiothreitol treatment [ 141,su chromatin digestion resulted in yield of active complex rather than a stimulation of existing activity experiments, the addition of ei or deoxyribonuclease individually to dithiothreitol-treated sperm head preparations did not lead to increased activity in the 1, I5 g/ ml density region. The results, therefore, are most probably due to the combined specific proteolytic and nucleolytic enzymatic activities and not to non-specific effects. In-

50

Witkin and Bendich

Fig. 1. Abscissa: fraction; ordinate: (left) cpm (0-O); (right) density (g/ml, X---X). The effect of trypsin and deoxyribonuclease on the release of endogenous DNA polymerase activity from human sperm heads. Sperm heads were purified from two eiaculates and susvended by gentle homogenization in 1 ml of 0.02 M dithiothreitoi, 0.05 M T&J-HCI, vH 7.5, and incubated at 0°C for 90 min. The samvle, now containing swollen sperm heads, was next divided into two eaual Darts. Trvvsin (100 ~alml) was added to one part &d both Sam&s incubated at 37°C. After 5 min. 200 &ml sovbean trvvsin inhibitor. 10 ualml deoxyribonuzease and 2 nrM Mg2+ were .added to the trypsin-containing sample and both samples incubated at 37°C for an add~tiqnal 15 min. The two samples were next centrifuaed for 10 min at 10000 a and the supematants layered onto linear 5 ml 15d% (w/w) sucrose gradients in 0.05 M Tris HCl, pH 7.5,0.005 M dithiothreitol, 0.002 M EDTA for centrifugation in an SW 50L swinainn bucket rotor for 16 h at 165000 P. Fractions we& collected from below, density (X---G) determined from refractive indices, and ahquots assayed for endogenous DNA polymerase activity (see Methods).

cubation of the complex with ribonuclease, in contrast to the results with deoxyribonuclease, led to the loss of polymerizing activity [13] (see below). The release of an endogenous DNA-synthesizing complex from swollen sperm nuclei from which all outer membranes had been stripped, and its increased yield following chromatin dissolution strongly argue that the complex is located within the sperm head, in intimate Exp Cell Res 106 (1977)

association with the cross-linked chromatin. The DNA-synthesizing complex can also be purified from whole sperm, without the use of detergent. Washed sperm were suspended in 0.02 M dithiothreitol and treated with trypsin and deoxyribonuclease, as described above. This procedure resulted in the complete rupture of the sperm heads, while the tails remained intact. Sucrose banding of the 10000 g supernatant from this solution yielded most of the endogenous DNA polymerase activity at a peak density of 1.17 g/ml (fig. 2, top), as was the case with activity from purified sperm heads. Treatment of the 10000 g supernatant with 1.5 % Sarkosyl for 20 min at 4°C prior to banding in sucrose resulted in a shift in endogenous DNA polymerase activity to a density of 1.20-1.23 g/ml (fig. 2, bottom). Similar increases in density were observed when the 10000 g supernatant from dithiothreitol-treated purified sperm heads was incubated with phospholipase C and ether (data not shown). The DNA synthesizing complex thus appears to possess a lipid-containing component. The resistance of the DNA polymerase activity to trypsin digestion might thus be due to its enclosure within a protease-inaccessible structure. Characterization of the endogenous polymerization product

Peak fractions from a sucrose gradient of banded sperm DNA-synthesizing complexes were pooled, centrifuged, the pellets resuspended in 0.05 M Tris-HCl, pH 7.5, 0.02 M dithiothreitol, and divided into two equal fractions. MgClz and NaCl were added to both, and one member of each received 100 pg/ml ribonuclease 1. Following 30 min at 22°C dATP, dCTP, dGTP, actinomycin D, and [3H]dTTP were added, and the samples incubated at 37°C for 10

Human sperm DNA-synthesizing

E

-5

--

-

3

10

-

Fig. 2. Abscissa: fraction; ordinate: (right) density (g/ml, A---A).

3

! (left) cpm (O-O);

Release of endogenous DNA polymerase activity from intact human sperm. Sperm from a single eiaculate were pelleted 6y centrgugation at 3 000 g for”10 min, resuswnded in phosphate-buffered saline r131 and subjecied to three ad;litional cycles of ce&i: funation and resuspension. The nellet was then suspended by gentle homogenization in 1 ml 0.05 M Tris-HCl. PH 7.5. 0.02 M dithiothreitol and incubated at 0°C for-90 min. The sperm were then treated with trypsin and deoxyribonuclease and a low speed supernatant obtained, as described in fig. 1. The sample was then divided into two equal aliquots, Sarkosyl (1.5 %) added to one part>.and both kept at 0°C for 20 rn& The samples were then centrifuged at 10000 g for 10 min and the supematants centrifuged in equilibrium sucrose gradients for the determination of endogenous DNA polymerase activity, as in fig. 1, except for the inclusion in the assay mixture of 0.01 %I Triton X-100.

min. Nucleic acids were extracted from the reaction mixture with sodium dodecyl sulfate-phenol, centrifuged at 200000 g for 90 min through a lo-30% glycerol gradient, fractionated and acid-insoluble radioactivity determined. The [3H]DNA product, still associated with its endogenous template, was heterogeneous in size; 60% of the radioactivity was located in the bottom half of the gradient (fig. 3). In contrast, pretreatment of the DNA-synthesizing complex with ribonuclease resulted in the appearance of radioactivity only in the low molecular weight region (fig. 3). These results indicate that the majority of the [3H]DNA syn-

complex

51

thesized in the reaction without ribonuclease was associated with high molecular weight RNA. The low molecular weight [3H]DNA was either not RNA-associated or might represent end-addition of [3H]dTMP on ribonuclease-sheared RNA fragments. We have previously demonstrated a ribonuclease-enhanced polymerization of c3H]TMP by a fraction derived from sperm heads and speculated that residual semen phosphatase may convert RNA fragments to functional primers [13]. When the synthetic primer dT,, was added to an endsgenous sperm-derived reaction, a Bow molecular weight product resulted that was analogous to that obtained after ribonu-

80.

60.

40.

70

2c X:13,'

Fig. 3. Abscissu: fraction;ordinate:

-i, : 1::

cpm. Velocity centrifugation of sperm-derived endogenous product-template complexes. Particles from the sperm in two ejaculates having a density in sucrose of 1.15-1.19 &nl. were susaended in 0.05 M Tris-HCl. pH 7.5 ~1% O.b2 M dithTothreito1, each divided into two equal parts, incubated for 30 min in 7.5 pmoles M&l,. 10 wmoles NaCl in either the absence (03) orpresenck (O-O) of 100 &ml pancreatic ribonuclease i, c&led at tic, diluted to a final volume of 0.5 ml with 80 nmoles each dATP, dCW, and dGTP, 40 tie actinomvcin D and 100 Ki T3HldTTP (15.7 Ci/ mmoie) and iicubated at 37”b for 16 min. The endogenous “H product-template nucleic acid complexes were purified by extraction with sodium dodecyl sulfate-phenol and-centrifuged through IO-70 ‘% glycerol gradients at 200000 g for 90 min (see Methods). Fractions were collected by puncturing the bottom of the tube and acid-insoluble radioactivity measured on 50 ~1 aliquots.

52

Witkin and Bendich

Fig. 4. Abscissa: fraction; ordinate: (left) cp& (O-O); (right) density (g/ml, X---X). C&SO4 gradient analysis of the sperm-derived endogenous DNA polymerase reaction. Complexes from the sperm of three ejaculates, having a sucrose buoyant density of 1.15-l. 19 g/ml, were suspended in 0.05 M Tris-HCl, 0.02 M dithiothreitol, preincubated at 22°C with or without ribonuclease, utilized to synthesize au endogenous r3H]DNA product and the product-template complex purified, all as described in Methods. The sample prepared in the absence of ribonuclease was divided in two equal parts, one of which was heated at 100°C for 10 min and quickly cooled. All three samples were centrifuged to equilibrium in Cs2S0, gradients (86000 g, 65 h; WC), fractionated from below, density (X---X) determined from refractive indices, and acid-insoluble radioactivity measured. (a) Endogenous; (b) lOO”C, 10 min; (c) ribonuclease.

clease treatment (data not shown); this again suggests the complex contains a primer-dependent activity that is separable from the DNA polymerase. The yield of high molecular weight product was not enhanced by the addition of dT,,. Buoyant density of the endogenous product and template The ribonuclease sensitivity of the high molecular weight endogenous reaction product might arise from these possibilities: RNA might be a required primer for RNAor DNA-directed DNA synthesis, or a template for RNA-directed DNA synthesis. These alternatives can be distinguished by examining the behavior of the reaction product on isopycnic C&30, gradients [ 19, 201. The r3H]DNA product of an RNA tumor virus endogenous reaction bands as RNA in Cs2S04 since the DNA is noncovalently associated with a much larger ILxp CeilRes

IO6 (1977)

70s RNA template; physical removal of the template causes the product to band as DNA [21]. In contrast, where RNA is a primer for DNA-directed synthesis, heat denaturation of the 3H product-template complex does not result in a shift in density of the radioactivity from the RNA to DNA region because of covalent attachment [22]. Fractions in the 1.15-1.19 g/ml buoyant density region from a sucrose gradient of complexes obtained from three ejaculates were pooled, concentrated, and one-third of the sample removed and incubated with ribonuclease as described above. Both samples were then utilized to synthesize C3H]DNA in a 10 min endogenous reaction containing actinomycin D. Nucleic acids were purified from the reaction mixture by sodium dodecyl sulfate-phenol extraction, precipitated with ethanol and the products redissolved in 0.01 M Tris-HCl, pH 7.5, 0.002 M EDTA. The product derived from the sample not treated with ribonuclease was divided into two equal parts, one of which was subsequently heated at 100°Cfor 10 min, and all three samples centrifuged to equilibrium in C&SO, gradients. The results can be seen in fig. 4. In the unheated sample, three peaks of [3H]DNA were observed, one in the RNA region (density 1.70 g/ml), one at the density of RNA :DNA hybrids (1.55 g/ml), and one in the DNA region (density 1.45 g/ml). Heat denaturation, a procedure which would effect the separation of the r3H]DNA product from its endogenous template, resulted in a lowering of the buoyant density of most of the radioactivity to that of DNA, with a small fraction of the radioactivity remaining at the original density. Very little acid-insoluble product was evident in the ribonucleasetreated sample; the small amount present had a density intermediate between that of DNA and RNA, adding further support to

Human sperm DNA-synthesizing

the contention that ribonuclease-enhanced synthesis consists of terminal addition onto small RNA fragments. Additional Cs,SO, gradient analyses of the [3H]DNA products isolated from velocity centrifugations such as in fig. 3 demonstrated that the high molecular weight ribonuclease-sensitive product banded partly in the RNA region and partly as DNA; the low molecular weight ribonuclease-enhanced product banded at a density slightly higher than DNA (data not shown). The data are consistent with the interpretation that DNA is synthesized from an RNA template and that a portion of the [3H]DNA product is also covalently attached to an RNA primer. Conclusive evidence as to the nature of the endogenous template must await isolation of the particle nucleic acid.

DISCUSSION Tt is intriguing to speculate on the possible function of this sperm endogenous DNA synthesizing complex and to the similarities to the sequential progression of events that occur in embryogenesis or cancer. Parthenogenetically activated mammalian ova can develop into morphologically normal blastocysts, but, unlike those which form after fertilization, these die at the time when implantation in the uterus should occur or immediately thereafter [23]. Contributions from the male gamete thus appear to be necessary for attachment and for further development of the zygote at this stage, just prior to trophoblast migration and placenta formation. The major characteristics of this developmental stage: polyploidization and syncytium formation [24, 2.51,the invasion of trophoblast cells into the uterine epithelium, the transformation of maternal

complex

53

fibroblasts into decidua cells and penetration of maternal blood vessels [26], all parallel processes that are also seen in carcinogenesis. The genetic information coding for these events is obviously the result of normal DNA function. Induction, activation and expression of these genes in adult somatic cells following the intervention of chemical or physical agents, or the direct introduction of similar information via infection by exogenous virus or spermatozoa could thus lead to an anachronistic reiteration of some of the programmed sequence of events expressed during post-impiantation embryogenesis, giving rise to oncogenie alterations. The human sperm nucleus-derived synthesizing complex shares many properties with the cytoplasmic-derived particulate RNA-instructed DNA polymerase complex isolable from human tumors: a buoyant density of 1.15-l 018 g/ml [27, 28, 291, a shift to a density of 1.21-1.25 g/ml following detergent or phospholipase treatment [29, 301, ribonuclease-sensitivity of the high molecular weight endogenous [“ET]DNA product [31], and buoyant density of the endogenous 3H product-template complex [29, 31, 321. In common with the human tumor activity, no virus-like components have been observed in our sperm preparations by electron microscopy [15]. We have recently demonstrated that normal somatic cells can be penetrated by sperm in vitro; the subsequent appearance of morphological abnormalities and fetal antigens is reminiscent of malignant transformation [ 14, 33-351. The possibihty that the endogenous sperm complex plays a role in these processes is under investigation. We thank Dr E. Borenfreund and Dr P. J. Higgins for discussions. This work was supported in part by USPHS grant CA 08748 from the NCI, and the NCI, contract no. Not-CB43904. Exp Cell Rrs 106 (1977)

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19. Wu, A M & Gallo, R C, CRC crit rev biochem 3 (1975) 289. 20. Spiegelman, S, Bumy, A, Das, M R, Keydar, J, Schlom, J, Travnicek, M & Watson, K, Nature 227 (1970) 563. 21. Verma, I M, Menth, N L, Bromfeld, E, Manly, K &Baltimore, D, Nature new bio1233 (1971) 131. 22. Reitz, M S, Smith, P L, Roseberry, E A & Gallo, R C, Biochem biophys res commun 57 (1973) 934. 23. Graham, C F, Biol rev 49 (1974) 399. 24. Barlow, P R & Sherman, M 1, J embrvol - exu_ morph01 27 (1972) 447. 25. Sherman. M I. McLaren. A & Walker. P M B. Nature new biol238 (1972) 175. ’ 26. Larsen, J F, Physiology and genetics of reproduction (ed E M Coutinho & F Fuchs) part B, p. 287. Plenum Press, New York (1974). 27. Schlom, J, Spiegelman, S &Moore, D, Nature 231 (1971) 97. 28. Spiegelman, S, Cancer chemotherapy repts 58 (1974) 595. 29. Witkin, S S, Ohno, T & Spiegelman, S, Proc natl acad sci US 72 (1975)4133. 30. Feldman, S, Schlom, J & Spiegelman, S, Proc natl acad sci US 70 (1973) 1976. 31. Schlom, J & Spiegelman, S, Science 174 (1971) 840. 32. Gulati, S C, Axel, R & Spiegelman, S, Proc natl acad sci US 69 (1972) 2020. 33. Bendich, A, Borenfreund, E & Stemberg, S S, Science 183 (1974) 857. 34. Bendich, A, Borenfreund, E &Beju, D, Acta cytol 18 (1974) 544. 35. Higgins, P J, Borenfreund, E & Bendich, A, Nature 257 (1975) 488. Keceived November 16, 1976 Accepted December I, 1976