DEVELOPMENTAL BIOLOGY 43, 33%339(1975)

BRIEF NOTES

Cytochemical Studies on the Protamine-Type Protein Transition

in Sperm Nuclei after Fertilization and the Early Embryonic

Histones of Urechis caupo

NIRMAL K. DAS, JULIE MICOU-EASTWOOD, AND MAX ALFERT

Department of Cell Biology, College of Medicine, University o/Kentucky, Lexington, Kentucky 40506 (N.K.D.) and Department of Zoology, Uniuersity of California at Berkeley, California 94720

Accepted November 13, 1974

Cytochemical staining characteristics of nuclear histones during postfertilization maturation division and various early embryonic stages in Urechis have been studied. The transition of protamine-type protein to adult histones in the sperm nucleus is accomplished by 15 min after entrance into the egg cytoplasm. Newly synthesized egg proteins migrate into enlarging male and female pronuclei after this transition, followed by pronuclear DNA synthesis and fusion. The shift from protamine-type protein to adult histones, which occurs in the absence of RNA synthesis during the postfertilization maturation division of the egg, may be one of the processes involved in the normal structural reorganization of chromosomes. Such a reorganization is likely to be a prerequisite for chromosome replication and mitosis. No qualitative differences are detected in the stainability of histones of unfertilized eggs and embryos at the cleavage and later stages of development.

INTRODUCTION

Patterns of histone synthesis during early embryogenesis in several organisms have been studied by various workers in an attempt to elucidate the regulatory mech- anisms of embryonic differentiation (9, 15, 23). It has been suggested in some recent studies that the interaction between his- tones and nonhistone proteins may be involved in the sequential activation of genes during embryonic development (14, 19). A transition from somatic histones to protamine or arginine-rich histones occurs during spermiogenesis in various orga- nisms (2, 6). The significance of such a transition still remains unknown (7). How- ever, it is apparent that a shift of the highly basic proteins of the sperm nucleus back to somatic or adult histones is required for the normal embryonic development. Prota- mine or arginine-rich histones are no longer detectable after fertilization. These pro- teins are thought to be replaced in some

organisms, first by the so-called “cleavage” or “juvenile” histones (6, 7, 10); the transi- tion to adult histones is reported to occur at gastrulation (6, 7, 15). Other studies, however, suggest that the shift to adult histones takes place during or prior to early cleavage stages (3, 26).

We have studied histone patterns during early embryogenesis in Urechis caupo, a marine echiuroid worm. Unfertilized eggs of Urechis are at the diakinesis stage at the time of sperm entry (11). It takes about 1 hr, at 16-17”C, to complete the maturation division after insemination. The male and female pronuclei fuse about 15-20 min later. This organism provides an opportu- nity to study the transition of the prota- mine-type protein of the sperm nucleus (12) during a relatively extended period of time between sperm entry and fusion of pronuclei. It is of further interest to see whether cytochemical staining characteris- tics of histones during early embryogenesis

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334 DEVELOPMENTAL BIOLOGY VOLUME 43.1975

in this invertebrate, with some primitive early developmental patterns (21), are sim- ilar to those reported for other organisms (3, 6, 10, 26).

MATERIALS AND METHODS

Eggs from freshly collected Urechis caupo were fertilized in sea water at 1617°C (11). Samples were fixed over- night in 10% neutral formalin at various times, beginning 5 min and up to 24 hr after fertilization. They were washed sev- eral hours in distilled water to remove formalin, dehydrated and embedded in paraffin. Ten to 12 micron sections were stained with alkaline fast green (AFG) or bromphenol blue (BPB) techniques spe- cific for histones (1, 6). Some slides were subjected to acetylation procedures prior to staining (6). Autoradiographic techniques were used to detect the time of migration of labeled egg proteins into the sperm nucleus and DNA synthesis in both pronuclei. Immediately after fertilization, eggs were exposed to a mixture of tritiated phenylal- anine, leucine, and valine (50 &i/ml of each) or 3H-thymidine (40 &i/ml) alone. Samples were fixed in 10% formalin or acetic alcohol (1:3). Autoradiographs (NTB2 emulsion) of 5 pm thick paraffin sections were made and the time when both male and female nuclei become la- beled was determined.

RESULTS

When formalin-fixed sperm preparations are stained with AFG (l), only acrosomes give a positive reaction (Fig. 1); sperm nuclei which contain protamine-type pro- tein do not stain (12), since such proteins are labile in hot trichloroacetic acid (TCA) used to extract DNA in this staining proce- dure (2). These nuclei stain with BPB technique in which DNA is extracted by the mild picric acid hydrolysis (6). The nuclei of sperm which remain attached to the egg surface 5 minutes after insemina- tion also do not stain with AFG. About 7 min after insemination the sperm nucleus

is seen inside the egg cytoplasm (see also 21); such a nucleus begins to stain lightly with AFG (Fig. 2). Ten to 15 min after insemination sperm nucleistains well with AFG, as do meiotic chromosomes of unfer- tilized and fertilized eggs and both pronu- clei at fusion (Figs. 3-6). Nuclei of embryos from the early cleavage to later stages of development also give positive reaction to AFG or BPB (Figs. 7-10). The relatively large interphase nuclei of early cleavage cells stain faintly, as compared to inter- phase nuclei of embryos at later develop- mental stages and mitotic chromosomes of all embryonic stages (compare Fig. 7 with Figs. 8, 10). Acetylation blocks AFG and BPB stainability during all stages of devel- opment (Figs. llA, 11B).

Autoradiographs of sections of fertilized eggs labeled with a mixture of tritiated amino acids for 50 min show an absence of label in the relatively condensed male nucleus (Fig. 12). Such nuclei, as men- tioned above, stain well with AFG (Fig. 5). Labeled proteins appear in the male and female nuclei at about 75 min after fertili- zation when these nuclei are increasing in size (Fig. 13). Autoradiographs of eggs labeled with 3H-thymidine after fertiliza- tion show that DNA synthesis begins in such enlarged haploid nuclei (Fig. 14); synthesis may continue during the process of fusion of these nuclei. Autoradiographs further reveal that all male nuclei in poly- spermic eggs undergo synchronous DNA synthesis (Fig. 15); similar observations were also made on a sea urchin (5).

DISCUSSION

The ability of the sperm nucleus to stain with AFG soon after its entrance into the egg cytoplasm and the loss of this staina- bility resulting from acetylation strongly suggest that the shift from the protamine- type protein to histones occurs early during embryogenesis in Urechis. It is not known whether the male nucleus acquires all major histones during the post fertilization maturation division; no biochemical analy-

FIGS. l-11. Air-dried sperm preparation and sections (10 orn) of fertilized eggs of Urechis stained with alkaline fast green (AFG) and bromphenol blue (BPB). x 800.

FIG. 1. Sperm preparation; only acrosomes stain with AFG. FIGS. 2-5. Seven (Fig. 2), 10 (Fig. 3), 15 (Fig. 4), and 48 (Fig. 5) min after fertilization: sperm nucleus (N)

stains lightly with AFG, 7 min after fertilization; sperm nuclei in other samples (Figs. 3-5) and meiotic chromosomes (arrow) in all samples (Figs. 2-5) are well stained with AFG. The sperm nucleus in Fig. 2 was first identified by trichloroacetic acid-Feulgen staining (6).

FE. 6. Seventy-five min after fertilization; both male and female pronuclei (in the process of fusion) are AFG positive.

22.5

FIGS. 7-9. One hundred ten min after fertilization; condensed meiotic chromosomes (Fig. 8) stain more intensely with AFG than diffuse chromatin of interphase nuclei (Fig. 7) at the Z-cell stage. Condensed mitotic chromosomes also stain well with BPB (Fig. 9).

FIG. 10. Five and one-half hr after fertilization; nuclei of the 64-cell stage embryo stained with AFG. FIGS. 11A and IIB. Forty-eight min after fertilization; egg section stained with hematoxylin (Fig. 11A) to

show the male nucleus (N) and second meiotic chromosomes (CHR); the same section stained with AFG after acetylation (Fig. 11B). Note that the male nucleus and memtir chromosomes lose AFG stainability after acetylation (compare Figs. 11A and 11B). Hematoxylin stain IS lost during hot trichloroacetic acid hydrolysis used to extract DNA. Arrows point to the same dirt spot on the section.

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BRIEF NOTES 337

FIGS. 12 and 13. Autoradiographs of sections (5 pm) of eggs exposed to tritiated phenylalanine, leucine, and valine (50 pCi/ml of each) for 50 (Fig. 12) and 75 (Fig. 13) minutes following fertilization. Note that the male nucleus (arrow) in Fig. 12 is unlabeled, while both pronuclei (male and female undistinguishable) in Fig. 13 are labeled. Exposure time 25 days, hematoxylin stain. x 800.

FIGS. 14 and 15. Autoradiographs of sections (5 pm) of eggs exposed to 3H-thymidine (40 rCi/ml) for 65 min after fertilization. Both male and female nuclei (undistinguishable) are labeled (arrows). The presence of relatively large number of grains in the cytoplasm probably indicates mitochondrial DNA synthesis. Exposure time 70 days. hematoxylin stain. x 1400.

sis of such nuclei has yet been possible. Gel larity to the protamine-type proteins of electrophoretic analysis (unpublished), some lower marine organisms (12). however, shows that the sperm nucleus The transition of protamine-type protein contains a single protein before its entrance to adult histones in the male nucleus takes into the egg cytoplasm. The amino acid place during the period of postfertilization composition of this protein reveals its simi- maturation division inactive in RNA syn-

338 DEVELOPMENTAL BIOLOGY VOLUME 43,1975

thesis (11). This may indicate that mater- nal messenger RNA is available for transla- tion during this period. Skoultchis and Gross (24) have demonstrated recently the presence of maternal histone messenger in a sea urchin. It is also possible that eggs already contain some histones, which are used during early embryogenesis. We have not detected migration of labeled egg pro- teins into the sperm nucleus at the time when it begins to give a positive reaction with AFG. It is possible that histones made during this time may not have been suffi- ciently labeled for autoradiographic detec- tion. However, preincubation of eggs with tritiated amino acids, 20 hr prior to fertili- zation, also fails to show the presence of labeled proteins in sperm nuclei during postfertilization maturation division.

The present cytochemical results do not provide any evidence for the presence of special “cleavage” or “juvenile” histones (6, 10) in Urechis. The nuclei of interphase cells of early cleavage stages stain faintly with AFG or BPB, as compared to chromo- somes of mitotic cells at these stages. Similar results were also reported for other materials (16, 20). The diffuse state of chromatin in such relatively large nuclei could well account for this light stainabil- ity (16). Alfert (3) observed that the male pronuclei and also interphase nuclei of early cleavage cells of the mouse do not stain with AFG; it was suggested that this could be due to the interference by nonhis- tone proteins, rather than to lack of his- tones. Our preliminary polyacrylamide gel electrophoresis studies of acid soluble nu- clear proteins show no qualitative differ- ences in most major histones between un- fertilized eggs and embryos at cleavage, blastula and gastrula stages. The absence of such differences has also been noted in sea urchins (14, 23), amphibians (9, 13), and chicken (15).

The following sequence of events occurs in steps after fertilization in Urechis: Tran- sition of protamine-type protein to histones

in the sperm nucleus, entrance of newly synthesized egg proteins (perhaps nonhis- tone proteins) into the male and female pronuclei, initiation of DNA synthesis in haploid pronuclei, pronuclear fusion, mito- sis and cytokinesis (first cleavage division). The shift to adult histones, which occurs in the absence of RNA synthesis, is likely to be one of the processes involved in the normal structural reorganization of chro- mosomes. Such a reorganization may be necessary for chromosome replication and mitosis. Kiefer et al. (17) suggested that histone synthesis during early cleavage stages in the sea urchin is necessary for normal mitotic behavior of chromosomes. DNA synthesis in the haploid pronuclei of Urechis begins during or immediately after the entrance of some newly synthesized egg proteins, indicating that an interaction between adult histones and nonhistone egg proteins may be involved in the initiation of this process (see also 25). The lack of qualitative differences in the stainability of histones at various early developmental stages in Urechis would seem to argue against the involvement of histones alone in the control of patterns of RNA synthesis during embryogenesis. Recently Byrd and Kasinsky (9; see also 13) observed no qualitative changes in major histones syn- thesized during the cleavage and swim- ming stages in Xenopus laevis; patterns of RNA synthesis at these stages are very different (8). The specificity of gene ex- pression during embryonic development may involve biochemical modification of histones (4, 18) and/or their interaction with acidic nuclear proteins (14, 19, 22). Such proposals, however, need further ex- perimental verification.

This work was supported by General Research Support Grant No. RR 05373 from General Research Support Branch, Division of Research Facilities and Resources, N.I.H. and by a Grant No. GB 297741 from the National Science Foundation. We thank Miss Ann Behrmann and Mrs. Carolyn Barker for able technical assistance.

BRIEF NOTES 339

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