Changes in nuclear proteins of rat testis cells separated by velocity sedimentation.

The technique of velocity sedimentation at unit gravity has been used to separate rat testis cell suspensions into fractions enriched in particular cell types. Changes in the nuclear proteins from the various fractions have been characterized by polyacrylamide gel electrophoresis, and correlated with the changing morphology of the nucleus during spermatogenesis. The most striking alterations in both protein composition and nuclear morphology occur during spermatid maturation as both histone and non-histone proteins are replaced by highly basic, low molecular weight, spermatidal proteins. This replacement process is accompanied by a quantitative reduction in both histone and non-histone proteins. The synthesis of at least three basic proteins has been identified with late stage spermatids. One of these proteins is a highly basic sperm-specific protein containing high levels of cyst(e)ine and arginine. A second protein synthesized in late stage spermatids is lysine rich, while the third protein contains cyst(e)ine and co-migrates with histone F2a1 on acid-urea polyacrylamide gels. The changes in protein composition of rat testis nuclei after irradiation or hypophysectomy reflect the resulting changes in the cellular composition of the testis. After selective elimination of the germinal cells by irradiation, the electrophoretic pattern of acid-soluble proteins from the testis is very similar to that of somatic tissue. Thus, the cellular specificity of nuclear proteins demonstrated here using cell separation techniques is also apparent following treatments which selectively alter the cellular composition of the testis.

at unit gravity has been used to separate rat testis cell suspensions into fractions enriched in particular cell types. Changes in the nuclear proteins from the various fractions have been characterized by polyacrylamide gel electrophoresis, and correlated with the changing morphology of the nucleus during spermatogenesis. The most striking alterations in both protein composition and nuclear morphology occur during spermatid maturation as both histone and non-histone proteins are replaced by highly basic, low molecular weight, spermatidal proteins. This replacement process is accompanied by a quantitative reduction in both histone and non-histone proteins. The synthesis of at least three basic proteins has been identified with late stage spermatids.
One of these proteins is a highly basic sperm-specific protein containing high levels of cyst(e)ine and arginine. A second protein synthesized in late stage spermatids is lysine rich, while the third protein contains cyst(e)ine and co-migrates with histone FZal on acidurea polyacrylamide gels. The changes in protein composition of rat testis nuclei after irradiation or hypophysectomy reflect the resulting changes in the cellular composition of the testis. After selective elimination of the germinal cells by irradiation, the electrophoretic pattern of acid-soluble proteins from the testis is very similar to that of somatic tissue. Thus, the cellular specificity of nuclear proteins demonstrated here using cell separation techniques is also apparent following treatments which selectively alter the cellular composition of the testis.
Spermatogenesis involves a progressive differentiation of the cells of the seminiferous epithelium toward a terminal stage represented by the mature spermatozoan (1). The process begins with a period of proliferation in which spermatogonial cells divide several times to augment the number of potential germ cells. Most of these cells enter a period of growth as spermato-* This work was supported by United States Public Health Service Grants HD-05803 and CA-06294, Contract FI)A-73-204 from National Center for Toxicological Research, Robert A. Welch Foundation Grant G138, and a grant from the Population Council, New York. cytes, and each spermatocyte undergoes two meiotic divisions to produce haploid spermatids. This process concludes with a period of spermatid maturation, during which the nuclear material condenses to form the headpiece of a compact, motile spermatozoan, and most of the cytoplasm is shed as a residual body.
During these processes of proliferation, growth, and maturation, significant changes occur in the kinds and quantities of macromolecules synthesized by the cells (1,2). Of primary interest to us are mechanisms underlying the replacement of nuclear proteins by sperm-specific proteins, such as those described for trout by Dixon and co-workers (3). Arginine-rich proteins isolated from sperm of several mammalian species have been characterized (4)(5)(6). With the exception of mouse (6), all are rich in arginine and cyst(e)inc, and they contain 2 tyrosine residues, and have alanine as the NH*-terminal amino acid. A similar arginine-rich protein has been isolated from rat epididy-ma1 sperm and characterized by Kistler ef al. (7). In addition, another low molecular weight basic protein, similar to the protein isolated by Lam and Bruce (6) from mouse, was found in rat testis. This protein contains both lysine and arginine, but not cyst(e)ine, and may be replaced by the cyst(e)ine-containing, arginine-rich protein during the later stages of spermatid maturation (7,8). In addition to the process of nuclear protein replacement by specialized basic proteins, we are interested in the changes, properties, and functions of the non-histone nuclear proteins in spermatogenic cells. These proteins are of special interest because of their proposed role in the regulation of gene transcription (9). The hormone-dependent changes in the synthesis and phosphorylation of acidic chromatin proteins during maturation of the rat testis (10) are consistent with the proposed function of these proteins in the regulation of gene expression.
Since the adult mammalian testis contains a population of cells in the process of differentiating toward a specific cell type, spermatogenesis may be viewed as a model system for studying changes in nuclear proteins associated with cellular differentiation. The feasibility of using the mammalian testis as a source of differentiating cells for biochemical studies has been greatly enhanced by the development of techniques for the separation of testis cells into relatively homogeneous populations (11). The separation of testicular cells by velocity sedimentation at unit gravity provides fractions enriched in specific cell types, which can be identified with particular stages of spermatogenesis (ll-14). Further purification of specific stages may be achieved by a   25'% (26).

RESULTS
Fractions from the Staput were checked microscopically and pooled on the basis of composition into eight major fractions, designated 13 to I. The cellular compositions of these fractions are shown in Table I, with the predominant cell type in each fraction indicated by the boxes. Highly enriched preparations of pachytene spermatocytes (Fraction B), round spermatids (E and F), and late spermatids (H and I) were routinely obtained. The relative purity of these fractions is demonstrated by comparing the photomicrographs of cells in Fractions C, F, and H with a photomicrograph of a total testis cell suspension before separa-tion ( Fig. 1). Nuclei isolated from each fraction were examined by electron microscopy, and judged to be free of visible cytoplasmic contamination. Nuclei from fractions enriched in late spermatids, however, are generally contaminated by the acrosome, as well as by the ribonucleoprotein aggregates derived from the residual bodies, and by the flagella of spermatids. These contaminants can be eliminated from late stage spermatids by sonication2 As a further check for possible cytoplasmic contamination, [Wlarginine-labeled nuclei were isolated in the presence of cytoplasm (10,000 x g supernatant) from [3H]arginine-labeled testis and vice versa. The cross-contamination of nuclear proteins by cytoplasmic proteins in these experiments was less than 2%.
To determine the extent of non-chromatin-associated proteins remaining in the nuclei, chromatin was prepared from nuclei isolated by the described procedure, and the electrophoretic profile of chromatin non-histone proteins was compared with that of whole nuclei (Fig. 2). The close similarity in protein composition indicates that any contamination of nuclei by nonchromatin proteins does not contribute significantly to the protein pattern observed in polyacrylamide gels. Acid extraction of nuclei from the various Staput fractions removes histone and some nonhistone proteins. The electrophoretic patterns of the acid-soluble proteins from fractions representing different cell types are remarkably different (Fig.  3). For example the spermatidal proteins (Fig. 3, Bands 3 and 4), which are major components of the fraction enriched in late spermatids (Fig. 3g)  senting early stage cell types (Fig. 3, c and d) . Furthermore, the ratio of the 2 lysine-rich histones (Fig. 3, Bands 1 and 2) changes in moving from pachytene spermatocytes (Fig. 3c) to early spermatids (Fig. 3e).
Quantitation of individual histones from densitometric tracings of the gels indicates that the relative amounts of protein in each histone region are similar in the various fractions (Fig. 4A). The profile of [3H]arginine incorporation, however, indicates the synthesis of acid-soluble non-histone proteins predominantly in Fraction B, and a differential synthesis of the histones in different Staput fractions (Fig. 4B). In Fraction B, enriched in pachytene spermatocytes, major incorporation occurs in the region of histones F3, F2b, and F2a2. This synthesis is unlikely to be the result of spermatogonial contamination, since the specific activity of the proteins in this region is generally constant in all fractions, while the spermatogonia are most enriched in Fraction G (Table  II). When late pachytene spermatocyte nuclei were further purified by rerunning nuclei prepared from Fractions B and C through another Staput separation, a fraction which contained over 90% pachytene nuclei was obtained. The specific activity of the proteins in the F2b region was unchanged by this further purification of nuclei, indicating that histone synthesis is indeed occurring in late pachytene spermatocytes. In Fraction H (Fig.  4B), enriched in late spermatids, major incorporation of [3H]arginine is observed in the region of histone F2al. In Fraction I, enriched in later stage spermatids, most of the incorporation occurs in the rapidly migrating proteins, while a small but significant level of incorporation persists in the F2al region.
Comparison of the relative specific activities of individual histones from each Staput fraction (Table II) suggests that the F2al histone is selectively synthesized in later stages of spermatogenesis. When Fractions H and I were pooled and the isolated 5795 nuclei rerun on a second Staput in sucrose, the specific activity of the F2al region was enhanced compared with the starting material. The potential contribution of contaminating spermatogonia to the pattern of synthesis was ruled out, since the specific activity of histone F2al is highest in Fraction H, while spermatogonia, which are most active in DNA synthesis, are localized in Fraction G (Table II).
In order to identify the site of F2al synthesis more precisely, late spermatid nuclei (Steps 11 to 19) were prepared by sonication of whole testes. A sample composed of 89% sperm heads, 10% residual bodies, and 1.9% round nuclei was further purified by Staput separation in sucrose to provide a fraction which was 96% sperm heads, 4% residual bodies, and 0.3% round nuclei. The densitometric tracings and radioactivity profiles from these preparations before and after Staput separation (Fig. 5) indicate that the synthesis of an acid-soluble protein similar in electrophoretic mobility to F2al is localized in late stage spermatids.
To characterize further the nuclear proteins synthesized by late spermatids, sonication-resistant heads were prepared from testes following a 2-hour pulse with [35S]cystine and IaH]arginine. The radioactivity profile after electrophoretic separation of the acid-soluble proteins (Fig. 6) demonstrates that a cyst(e)inecontaining protein which migrates with histone F2al on acidurea polyacrylamide gels (20) is rapidly synthesized in late stage spermatids, and comprises a major protein component of sonication-resistant sperm heads. Although not apparent from the data shown, two closely migrating bands are evident in the F2al region of these gels. Fig. 6 also demonstrates the presence of the two rapidly migrating spermatidal proteins observed in Fig. 3. The slower migrating protein has a high arginine and cyst(e)ine content, while the faster migrating protein is high in lysine and arginine. The data in Fig. 5 and Table II suggest that synthesis of these spermatidal proteins is localized in the late spermatid cells. The close correlation between the presence of these proteins and the distribution of elongated spermatids (Steps 16 to 19) is shown in Fig. 7.
The amino acid composition of the two spermatidal proteins from sonication-resistant testicular sperm heads is shown in Table III. Allowing for slight contamination of these proteins by other proteins, the analyses are reasonably close to those reported by Kistler et al. (7) for a testis-specific protein, TP (Band 4), and a sperm-specific protein, Sl (Band 3).
The non-histone proteins (acid-insoluble residue) of rat testis nuclei are a complex and heterogeneous group, ranging in molecular weight (determined by sodium dodecyl sulfate-acrylamide gel electrophoresis) from 10,000 to 120,000 (Fig. 8). For purposes of discussion, the gel is divided into five regions. Against a background of proteins which are present in every cell fraction, many quantitative changes are consistently observed. and I), while at least two major proteins are present in all fractions. In Region V, one non-histone protein, which is absent or reduced in the early stages of spermatogenesis, appears as a major component of elongating spermatids (Fractions H and I (Fractions C and D). The pattern of non-histone proteins from spermatids prepared by sonication (Fig. 9) demonstrates that acid-insoluble proteins are still present in spermatids (Steps 11 to 19). These proteins were not detectable, however, in sperm heads from the epididymis after solubilization in guanidine HCl and acid extraction of the basic sperm protein.
In order to substantiate the data obtained by cell separation techniques, nuclear proteins were examined after irradiation or hypophysectomy, treatments which selectively alter the cellular composition of the testis (31-33). Seventy days after local irradiation of the testes with cobalt 60, cytological examination revealed only Sertoli, myoid, and interstitial cells present with no evidence of germinal cells. The electrophoretic pattern of the acid-soluble proteins from the irradiated testes (Fig. 10) is different from the normal control pattern in at least three ways. First, the slower migrating of the 2 lysine-rich histones (Band 1) is absent after irradiation.
Second, a change is observed in the density of the F2b region, presumably resulting from the absence of a protein which is similar to, but slower migrating than, histone F2b, and which is unique to the testis (34). Third, the spermatidal protein(s) are absent after irradiation.
As a result of these changes, the histone pattern after irradiation is very similar to that of liver, or any other somatic tissue. The pattern changes after hypophysectomy are somewhat less striking, and may reflect the presence of early stage germinal cells which remain in the testis after hypophysectomy (31).

DISCUSSION
The results presented here demonstrate several changes in the nuclear proteins of rat testis cells during spermatogenesis. We have used cell separation techniques to correlate these changes with cells in specific stages of spermatogenesis, and to obtain information regarding the differential synthesis of nuclear proteins by specific cell types. Our data indicate that both histone and non-histone proteins are progressively replaced by rapidly migrating basic proteins in the later stages of spermatid maturation. The synthesis and accumulation of these basic proteins  (21). The cellular composition of Fractions B through I before isolation of nuclei is given in Table I. C, D, F, G, H, and Z are Staput fractions from which non-histone nuclear proteins were isolated. T, trypsin control: whole testis cell suspension prepared with trypsin for Staput separation and held at 4" for 4 hours to simulate conditions used for Staput fractionation.
M, mechanical control: whole testis cell suspension prepared without trypsin under conditions designed to minimize protein degradation (see under "Materials and Methads"). Note that the non-histones of 7' and M are virtually identical, indicating no visible degradation of these proteins during preparation of the cell suspension using trypsin. Regions of the gel, Z to V, are designated on the left; molecular weight estimates of major proteins are indicated on the right. FIG. 9 (center). Sodium dodecyl sulfate-polyacrylamide slab gel showing non-histone proteins (acid-insoluble residue) after a correlation exists between the amount of non-histone protein associated with DNA and the level of RNA synthesis. Careful fractionation of the non-histones, combined with further studies on their synthesis and modification in rat testis cells, should help in identifying and characterizing individual proteins. It should then be possible to formulate and test meaningful hypotheses regarding the function of specific nuclear proteins in modifying the structure of chromosomes and in regulating the transcription of RNA.
--c--j --;+1 a b I ( -, :\ =F2b F2a2 \F2al acid extraction of sonication resistant spermatids. Mature spermatids were prepared from whole testes as described elsewhere.2 a, Non-histone proteins from fraction composed of 89% spermatids, 10% residual bodies, and 0.9% round nuclei. b, Non-histone proteins from spermatid fraction after being purified on Staput. Composition is 96% spermatids, 4y0 residual bodies, 0.3% round nuclei. Position of major protein bands is indicated. Densitometric scans of the acid-soluble proteins extracted from these spermatids are shown in Fig. 5. FIG. 10 (right). Electrophoretic patterns of acid-soluble nuclear proteins from rat testes after treatments to alter the cellular composition. Proteins were separated on polyacrylamide gels containing 2.5 M urea (20). RL, rat liver; ZR, proteins isolated 70 days after local irradiation of the testis with cobalt 60 to destroy all germinal cells; N, normal rat testis control; HX, proteins isolated 55 days after hypophysectomy.
Note that after irradiation (ZR) the pattern from testis resembles the somatic histone pattern represented by rat liver (RL). After irradiation and hypophysectomy, the spermatidal proteins are absent.