The acquisition of egg cytoplasmic non-histone proteins by nuclei during nuclear reprogramming

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Experimental Cell Research 108 (1977) 421-427

T H E A C Q U I S I T I O N OF EGG C Y T O P L A S M I C N O N - H I S T O N E P R O T E I N S BY N U C L E I D U R I N G N U C L E A R R E P R O G R A M M I N G NANCY J. HOFFNER and MARIE A. DiBERARDINO D e p a r t m e n t o f A n a t o m y , The Medical College o f Pennsylvania, Philadelphia, P A 19129, U S A

SUMMARY Inseminated eggs and nuclear transplants o f R a n a pipiens were labeled with [aH]tryptophan. Both the pronuclei of fertilized eggs and the late gastrula endodermal nuclei of transplants concentrated label during the first cell cycle of the egg, and this label was resistant to boiling TCA. These studies demonstrate that nuclear reprogramming is accompanied by the nuclear acquisition of cytoplasmic non-histone proteins from the egg.

Transplantations of amphibian nuclei into enucleated eggs have revealed the developmental potentiality of nuclei during the course of cellular differentiation. Since the initial experiments were conducted by Briggs & King [1], a great deal of evidence has accumulated from different laboratories on a variety of amphibians which demonstrates that many nuclei from cells of early embryos promote normal development of the test eggs, and are therefore functionally equivalent to the zygote nucleus. However, nuclei from more advanced developmental stages and more differentiated cells promote progressively more numerous and severe developmental abnormalities after transplantation into enucleated eggs [see 2-5]. Despite this severe decrease in nuclear potentialities, a small percentage of nuclei from adults are able to program enucleated eggs to develop into swimming larvae which contain the various cell types normally found in tadpoles [6-10]. However, even

with this extensive cell differentiation, these nuclear transplants rarely survive much beyond the early larval stages. Studies conducted on nuclei from cells which display specific phenotypes permit one to test the developmental potencies of nuclei of specialized cells rather than inadvertently testing nuclei of undetermined cells which can exist in a population of cells from differentiated tissues. The most extensive development obtained so far from nuclei of specialized cells has been from keratinized skin cells [11] and from immunoglobulin bearing lymphocytes [12]. A few of these nuclei promoted the test eggs to develop into early larvae possessing a variety of differentiated cell types. Although none of these nuclear transplants developed into feeding larvae, their extensive development and formation of various cell types has been interpreted to mean that nuclei of specialized cell types retain a full complement of genes capable of functioning normally in development [11]. Exp Cell Res 108 (1977)

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The nature of the restrictions of the many nuclei which do not promote development is still not understood. Some insight into this problem has come from two kinds of studies. On the one hand, it has been found that chromatid bridges and breaks arise during the first cleavage in most nuclear transplants derived from determined cells. As a result of these events, variable deletions occur in the chromosomes, and lead to variable abnormal development and ultimately are the cause of developmental arrest [4]. On the other hand, when the nuclear transplantations are conducted at a low temperature and spermine is included in the operation medium, there is a significant improvement in the percentage of larvae obtained from tail bud endodermal nuclei [13]. These two studies taken together indicate that there exists a temporal incompatibility between egg cytoplasm and chromosomal replication of nuclei from advanced cell types. This incompatibility can be overcome in some nuclei by extending the length of the first cleavage cycle with low temperature; the spermine might displace the histones bound to the DNA and thereby facilitate DNA replication. It is important to note that although most nuclei from advanced embryonic and adult cells do not promote normal development of the host eggs, most of these nuclei do undergo some important changes during the first cell cycle of the egg. For example, less than 1% of adult blood cell nuclei undergo DNA synthesis in situ, but when they are transferred to egg cytoplasm, 80% of the injected nuclei initiate DNA synthesis [14]. It is precisely during this process of nuclear reprogramming of the transplanted nucleus that egg cytoplasmic proteins (types unknown) enter the transplanted nucleus [15-16]. Our recent studies have shown that during the same period most Exp Cell Res 108 (•977)

[3H]tryptophan labeled nuclear non-histone proteins originally present in late grastrula endodermal nuclei in vivo leave the nuclei after transplantation into enucleated eggs, whereas nuclear proteins marked with [aH]lysine remain for the most part in the transplanted nucleus [17]. Thus, a reprogramming of the nucleus occurs to the extent that complex nucleocytoplasmic exchanges occur--some involving loss of proteins associated with determined endodermal nuclei and some involving the acquisition from the egg of cytoplasmic proteins which enter the transplanted nucleus and presumably serve to support the required events of the new cell cycle. In this paper we report additional studies on events occurring during nuclear reprogramming. Specifically, we have asked whether transplanted nuclei and pronuclei of fertilized eggs acquire cytoplasmic nonhistone proteins from the egg during nuclear reprogramming? We have concerned ourselves with pronuclei of fertilized eggs, because this is one of the best natural examples of reversal of specialization of nuclear function. Similarities between pronuclei and transplanted nuclei will serve to support the interpretations of an experimental system, like nuclear transplantation,. The studies reported below will show that during the first cell cycle of the frog egg both pronuclei and transplanted nuclei concentrate significant amounts of cytoplasmic non-histone proteins during the period of reprogramming.

MATERIALS AND METHODS These studies were conducted on eggs and embryos derived from the leopard frog, Rana pipiens. Ovulation was induced by intra-abdominal injection of a combination of pituitary glands and progesterone, Mature ova were artificially inseminated with sperm suspensions derived from macerated testes. Details of these original procedures developed by Rugh as well

N u c l e a r accumulation o f non-histone proteins as current modifications have been summarized previously [18-19]. Embryos were reared in dechlorinated tap water at 18-19°C and all experiments were conducted at this temperature. [5-aH] DL-Tryptophan with a specific activity of 25000 mCi/mmole was obtained from Schwarz Bio Research. A final pH 7.0 solution of 0.083/zCi of the amino acid, diluted with sterile Steinberg salt solution [20] to a final volume of 0.2/zl, was injected into eggs by means of calibrated glass micropipettes. Fertilized eggs were injected in the animal hemisphere near the equator from 35-83 min post insemination and fixed at three intervals, namely 100, 120 and 160 rain after insemination. The lengths of radioactive incubation were 50--65, 62-77 and 77-80 min for the 100, 120 and 160 rain groups. The basic procedure for amphibian nuclear translantation was that devised originally for R. pipiens ]. In the experiments reported below, late gastrula endodermal cells were used as donor cells for nuclear transplantation. Mature eggs were activated by injection of 1 or 2 endodermal nuclei. Thirty to 60 min after activation-injection of the nucleated eggs, [aH]tryptophan was injected into the animal hemisphere near the equator but on the side opposite the site of nuclear transplantation. The other conditions of the injection of radioactive tryptophan were identical to those used on fertilized eggs. All the nuclear transplants were fixed 120 min after activation-injection. Fertilized eggs and nuclear transplants were fixed in 10% formaldehyde, serially sectioned, bleached and processed for autoradiographic studies. The incorporation of [aH]tryptophan into the nuclei and cytolasm of cells demonstrates the localization of nonistone proteins (see controls). Since the normal site of protein synthesis is cytoplasmic [21], acquisition of radioactive non-histone proteins by nuclei would be due to migration of these non-histone proteins or their gubunits from the cytoplasm into the nuclei. Details concerning procedures for obtaining embryonic material, injection of labeled amino acids, nuclear transplantation, cytology and autoradiography have been delineated previously [17-19]. In order to determine whether pronuclei of fertilized ggg$ and transplanted endodermal nuclei concentrate cytoplasmic non-histone proteins, the number of grains in the volume of the nucleus was counted and compared with the grains present in equal volumes of cytolasm from four locations. The cytoplasmic counts ere averaged, all counts were corrected for back$round (average of four locations) and N/C ratios were determined. This approach is preferable to counting grains in a sample area of the nucleus and cytoplasm, since.it is possible that some grains may be located on the surface of the nucleus and not present within the nucleus. Amphibian oocytes near maturity and early embryos contain in their cytoplasm large quantities of yolk platelets. Our previous autoradiographic studies have shown that these yolk platelets contain very few grains following injection of [gH]tryptophan ([17]; this study; Matilsky, unpublished). Similarly, when I-labelled proteins are injected into Xenopus oocytes, very few grains are localized over the yolk platelets, and, therefore, these organelles are considered to be inaccessible

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cytoplasm and are excluded from the study [22]. In the sampled cytoplasm which we have analysed for radioactive tryptophan, we have included yolk platelets as part of the cytoplasmic volumes because exclusion of this organelle produces a bias. Yolk platelets in vitellogenic oocytes are not impermeable to certain precursor molecules or even some proteins [23]. Although yolk platelets of older oocytes and early embryos do not incorporate certain amino acids and proteins, this fact does not allow one to conclude that all substances fail to enter yolk platelets. In the case of nuclei, when these are injected into oocytes they do not incorporate [aH]thymidine; however, when they are transplanted to mature eggs, they do incorporate [gH]thymidine [24]. Thus, the physiological requirements and functions of cellular organelles can vary during their life history, but this fact does not support the proposal, that when certain activities are dormant, they should be excluded from a sample study. If this were so, we would have to consider all sub-microscopic elements in the cytoplasm as inaccessible if they fail to incorporate a certain tracer substance.

Controls Three series of controls were conducted. One series consisted of 618 inseminated eggs which were reared to the feeding larval stage. Ninety-six percent developed into normal larvae and therefore assured us that the quality of the material used for the experiments was optimal. The second series consisted of fertilized eggs which were injected with [gH]tryptophan (68 cases) or Steinberg salt solution (12 cases). Seventy-five percent of the tryptophan injected eggs and 100% of the eggs injected with salt solution developed into completely cleaved blastulae, indicating that normal cleavage was not significantly altered by the injection procedures. The third series comprised serially sectioned fertilized eggs and nuclear transplants which were exposed prior to autoradiographic processing to 5% boiling TCA for 15 min. This series did not display a reduction of grains compared with untreated autoradiograms, indicating that the [SH]tryptophan had been incorporated mainly into nonhistone proteins.

RESULTS F e r t i l i z e d eggs a n d n u c l e a r t r a n s p l a n t s w e r e f i x e d at a p p r o p r i a t e t i m e s d u r i n g t h e first cell c y c l e o f t h e e g g b e f o r e b r e a k d o w n o f the nuclear membrane. Thus, uptake of the l a b e l e d t r y p t o p h a n b y n u c l e i w o u l d h a v e to occur through the nuclear membrane.

I n s e m i n a t e d eggs An analysis of autoradiograms revealed that a l m o s t all o f t h e p r o n u c l e i o f f e r t i l i z e d eggs

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Table 1. Incorporation of [3H]tryptophan into egg cytoplasm and pronuclei Minutes Embryo no.

Post insemination

Length of radioactive incubation

126 127 126 125 128 129 111

100 100 100 100 100 100 120

61 57 61 65 53 50 71

111

120

71

110 109 112

120 120 120 120 160 160

74 77 66 62 77 88

113

119 116

Grain counts in autoradiogramsa Nucleus 17.0 39.8 14.3 4.0 17.5 79.3 12.5 8.0 49.3 67.8 86.0 53.5 62.5 >213.0

Cytoplasm

N/C

20.0 39.8 14.0 2.5 9.0 20.0 10.3 4.8 22.0 25.5 29.5 16.3 10.5

0.9 1.0 1.0 1.6 1.9 4.0 1.2

28.8

1.7

2.2 2.7 2,9 3,3

6,0 >7.4

a Grains present throughout the volume of each nucleus were counted, and the grains in four equal volumes of cytoplasm near each nucleus were determined and divided by four. All grain counts have been corrected foj background.

concentrated labeled tryptophan (table 1; fig. 1A). The few exceptions concerned 3 eggs fixed 100 min after insemination. In one case the number of grains in the nucleus was less than that present in an equal volume of cytoplasm (N/C 0.9), and in two cases the amount of grains was equal between the nucleus and an equivalent volume of cytoplasm (N/C 1.0). In those eggs fixed at 120 and 160 min after insemination, all the pronuclei displayed N/C values greater than 1. The length of radioactive incubation in the 100 and 120 min group extended from 50-65 min and 62-77 min, resulting in a variation of 15 min within each group. However, there is no correlation between the N/C ratios and the lengths of incubation within these two groups. F o r example, in a 100 min series, e m b r y o 129 with the shortest length of radioactive incubation had a high N/C ratio (4.0) and e m b r y o 125 with the longest length of incubation had a N/C Exp Cell Res 108 (1977)

ratio of 1.6, Similar relationships were obtained in the 120 min group. With the exception o f 2 embryos ( n o s . I 126 and 111) in the 100 and 120 min group, i respectively, all of the embryos were at a stage in the first cell cycle when the male and female pronuclei had fused. In those two cases in which the pronuclei had not yet fused, the two pronuclei within each egg were similar both in terms of absolute number of grains in the nucleus and cytoi plasm and N/C ratios. !

Nuclear transplants All of the nuclear transplants were fixed 120 min after activation o f the egg, yielding a series in which the transplanted nuclei resided in the cytoplasm of recipient eggs from 74--84 min. At approx. 130-145 min after activation of the host at 18°C, nuclear transplants have attained the metaphasd stage [4, 25]. F o r this reason nuclear trans~ plants were not fixed at a later interval

Nuclear accumulation of non-histone proteins

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O

la

Fig. 1. Nuclear accumulation of [aH]tryptophan from

the egg cytoplasm. (A) Pronuclei of fertilized egg no. 119, fixed 160 rain after insemination, N/C 6; (B) nuclear transplant no. 9, fixed 120 min after egg activation, N/C 2.5; (C) nuclear transplant no. 13, fixed

120 rain after egg activation, N/C 3.3. Large bodies with deeply stained centers are yolk platelets transferred from the cytoplasm of late gastrula endodermal cells.

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Table 2. Incorporation of [aH]tryptophan

into endodermal nuclei of transplants Nuclear transplant no. a

Length of radioactive incubation (min)

14 3 18 4 4 11 9 10 2 20 9 13 3

74 82 80 84 84 77 80 79 83 80 80 76 82

Grain counts in autoradiograms b Nucleus

Cytoplasm

N/C

10.5 26.3 33.3 70.3 46.3 25.5 73.8 55.8 15.5 98.0 78.3 35.3 44.3

9.0 17.3 17.8 36.0 22.5 10.3 29.3 21.8 5.8 34.0 26.0 10.8 12.0

1.2 1.5 1.9 2.0 2.1 2.5 2.5 2.6 2.7 2.9 3.0 3.3 3.7

a Transplants fixed 120 min post activation. Some transplants injected with 2 nuclei. b Grains present throughout the volume of each nucleus were counted, and the grains present in four equal volumes of cytoplasm near each nucleus were determined and divided by four. All grain counts have been corrected for background.

comparable to the fertilized eggs. In fact, the first cell cycle in nuclear transplants is about 30 min shorter than in fertilized eggs. Nuclear concentration of [SH]tryptophan occurred in all of the nuclear transplants examined (table 2; fig. 1 b, c). The N/C ratios ranged from 1.2 to 3.7. Within the narrow range of variable lengths of radioactive incubation (10 rain), there was no correlation between the lengths of incubation and the N/C ratios. Three nuclear transplants listed in table 2, each contained two transplanted endodermal nuclei (nos 3, 4 and 9). Both nuclei in no. 4 and no. 9 transplants had similar N/C values. However, in no. 3 transplant one nucleus had a N/C value of 1.5, whereas the other had a much higher N/C ratio of 3.7. The explanation of the latter is unknown at this time, but might be related to the fact that individual transplanted nuclei Exp Cell Res 108 (1977)

are known to respond differently to the egg cytoplasm. For example, transplanted nuclei swell and despiralize their heterochromatin to varying degrees [4] and also acquire [aH]thymidine in variable amounts [14]. These differences might reflect different states of nuclear differentiation and/ or perhaps be related to different intraperiods of the cell cycle of donor nuclei. DISCUSSION The present study has demonstrated in

R. pipiens that both pronuclei of fertilized eggs and endodermal nuclei transplanted int o eggs concentrate cytoplasmic nonhistone proteins during the first cell cycle of the egg. Thus, nuclear reprogramming is accompanied by the nuclear acquisition of egg cytoplasmic non-histone proteins in amounts 1.2-7 x greater than that present in equal volumes of sampled cytoplasm. It should be noted that the above studies involved the use of [aH]tryptophan as a marker for non-histone proteins. Thus, we are following only those non-histone proteins which were recently labelled in the egg cytoplasm. Other proteins could concentrate in the nuclei, e.g. newly synthesized histones, as well as previously synthesized histones and non-histone proteins~. but these would not be detected in our studies. Our present results together with previous studies [17] reveal that during the process of nuclear reprogramming of transplanted nuclei from determined endodermal cells, there is a bidirectional nucleo-cytoplasmic exchange o f non-histone proteins. The nature of these proteins at this time i~ unknown. One could speculate that thos~ egressing from transplanted nuclei includ~ non-histone proteins required for endodert mal determination; whereas those migrating [

Nuclear accumulation of non-histone proteins into the nuclei could include enzymes for unwinding chromatin, DNA polymerases and perhaps initiating protein(s) for DNA synthesis found to be present in high levels in the cytoplasm of eggs [26]. Future studies directed toward a characterization of these bidirectional non-histone proteins might elucidate whether some of these are involved in nuclear differentiation and whether some are concerned with nuclear reprogramming. We thank L. Artz and M. Matilsky for technical help and F. Linke for caring for the experimental animals, and appreciate the helpful comments on the manuscript provided by Drs R. Briggs, R. G. McKinnell and S. Subtelny. This investigation was aided in part by research grant GB-41838 from the NSF.

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8. Laskey, R A & Gurdon, J B, Nature 228 (1970) 1332. 9. DiBerardino, M A & Hoffner, N J, J exp zool 176 (1971) 61. 10. Kobel, H R, Brun, R B & Fischberg, M, J embryol exp morpho129 (1973) 539. 11. Gurdon, J B, Laskey, R A & Reeves, O R, J embryol exp morpho134 (1975) 93. 12. Wabl, M R, Brun, R B & DuPasquier, L, Science 190 (1975) 1310. 13. Hennen, S, Proc natl acad sci US 66 (1970) 630. 14. Graham, C F, Arms, K & Gurdon, J B, Dev biol 14 (1966) 349. 15. Merriam, RW, J cell sci 5 (1%9) 333. 16. Gurdon, J B, Results and problems in cell differentiation (ed J Reinert & H Holtzer) vol. 7, p. 123. Springer-Verlag, New York, Heidelberg, Berlin (1975). 17. DiBerardino, M A & Hoffner, N J, Exp cell res 94 (1975) 235. 18. DiBerardino, M A, Methods in developmental biology (ed F H Wilt & N K Wessells) p. 53. Crowell, New York (1967). 19. DiBerardino, M A, Hoffner, N J & Matilsky, M B, Methods in chromosomal protein research (ed G Stein, J Stein & L Kleinsmith) vol. 16, chap. 8, p. 141. Academic Press, New York & London (1977). 20. Steinberg, M, Carnegie inst Wash year book (report by J D Ebert) vol. 56, p. 347 (1957). 21. Goldstein, L, Advances in cell biology (ed D M Prescott, L Goldstein & E McConkey) vol. 1, p. 187. Appleton-Century-Crofts, New York (1970). 22. Bonner, W M, J cell bioi 64 (1975) 421. 23. Wallace, R A & Dumont, J N, J cell physioi 72, suppl. 1 (1%8) 73. 24. Gurdon, J B, Proc natl acad sci US 58 (1967) 545. 25. Subtelny, S & Bradt, C, J morphol 112 (1963) 45. 26. Benbow, R M & Ford, C C, Proc natl acad sci US 72 (1975) 2437. Received March 30, 1977 Accepted March 31, 1977

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