Changes in nuclear content of protein conjugate histone H2A-ubiquitin during rooster spermatogenesis

Electrophoretic analysis of acid-soluble chromosomal proteins isolated from rooster testis cell nuclei at different stages of spermatogenesis, revealed that the nuclear content of a protein identified by its solubility, electrophoretic mobility and amino acid analysis as the protein conjugate histone H2A-ubiquitin (uH2A, A24) changed markedly from meiotic cells to late spermatids. The protein was not detectable in tetraploid primary spermatocytes; it was present in 1.7% of the total amount of nucleosomal core histones in early spermatids and reached its maximum level (3.5% and 11%) at the end of spermiogenesis, when histones are replaced by the protamine galline.     

Studies on sex-organ development. Changes in nuclear and chromatin composition and genomic activity during spermatogenesis in the maturing rooster testis.

We developed a technique to separate nuclei of rooster testis by centrifugation through a discontinuous sucrose density gradient and by sedimentation at unit gravity. Four different major fractions obtained from testicular nuclei and one from the vas deferens were characterized according to their velocity of sedimentation, morphology and DNA content. The ratios (w/w) of basic proteins, non-histone proteins and RNA to DNA decreased during spermiogenesis both in nuclei and chromatin. Changes in the electrophoretic patterns of histones and non-histone proteins were detected especially in the elongated spermatids. The lack of uptake of [3H]uridine in elongating and elongated spermatids and in spermatozoa was demonstrated by radioautography and by the detection of labelled RNA extracted from different fractions of nuclei. Template activity for RNA synthesis and the binding of actinomycin D by testicular nuclei reached a peak in the elongated spermatid stage, when the histones are replaced by the protamine.     

Transformation of ram spermatid chromatin

In order to investigate the sequence of changes in nuclear basic proteins throughout ram spermiogenesis, we have used different techniques to obtain populations of spermatid nuclei in specific stages of maturation. Sedimentation of testis cells at 1 gravity followed by treatment with Triton X-100 resulted in one population of round spermatid nuclei (steps 1–a), one of non-round spermatid nuclei (steps 8b-15), and one of elongated spermatid nuclei (steps 12–15). Populations of non-round spermatid nuclei were obtained by treatment with EDTA (steps 9–15), by sonication (steps 12–15) and digestion by DNase (steps 13–15). Nuclear proteins, extracted either directly with dilute acid or following a reducing treatment with 2-mercaptoethanol were characterized by polyacrylamide gel electrophoresis.The most striking alterations in protein composition occur during the elongation phase (steps 8–12). The five histones are displaced from chromatin at the same rate. When they are freed of histones (step 12), the nuclei start to accumulate the sperm-specific protein (BNSP) which is then partly extractable by dilute acid without a thiol that is required for its extraction from more mature nuclei. This stepwise replacement process is accompanied by a reduction of the basic protein amount bound to DNA. As soon as histones begin to disappear, eight spermatidal protein fractions are present in the nuclei until the BNSP synthesis reaches its maximum rate. Except for one, they all contain cysteine and are partially intermolecularly cross-linked in the chromatin. After in vivo and in vitro labelling experiments, they are synthesized in elongating spermatids (steps 8–11). None are degradation products of histones.Correlations of the times of onset of EDTA, sonication and DNase resistances with changes in the basic nuclear proteins point out that stabilization and condensation of spermatid chromatin is promoted through a progressive increase in disulfide bridges.     

Partial characterization of a new basic nuclear protein from rat testis elongated spermatids.

A basic protein designated TP2 has been isolated from rat testis elongated spermatids. This new protein contains basic and acidic amino acids in relative amounts similar to those in histone F2al but is unusually rich in serine and proline. Techniques which were developed for preparing relatively homogeneous populations of spermatid nuclei were used to demonstrate that TP2 is most abundant and most actively synthesized in spermatids representing steps 12 through 15 of spermiogenesis.     

High mobility group proteins in ram spermatids

The four major high mobility group proteins HMG 1, 2, 14 and 17, HMG 19B and histone H1(0) were identified in the ram testis by their extraction and solubility characteristics and by their electrophoretic mobilities. HMG 14 and 17 were isolated by chromatography and amino acid analysis revealed that they were similar to their calf thymus analogues. A protein, named 2R and co-extracted with HMG 14, was also purified and analysed. Electrophoretic analyses of the proteins extracted by 0.75 M perchloric acid (PCA) or by 0.35 M NaCl from round and non-round spermatids, separated by centrifugal elutriation, showed that the four major HMG proteins disappear from nuclei in the oldest round spermatids, at the time the nuclear content of protein 2R and histone H1(0) increases in spermatids. Ubiquitin and HMG 19B were present in the round and elongating spermatids, but not in elongated spermatids which contained only protamine. The relation was considered between several protein changes and genetic inactivation and structural reorganization of the spermatid chromatin.     

Microelectrophoretic analysis of basic protein changes during spermiogenesis in the dogfish Scylliorhinus caniculus (L.)

Spermatogenesis in the dogfish is characterized by the synchronous development of germinal cells inside follicles. This particularity has permitted studies on precise stages of cell differentiation, especially on the evolution of chromatin structure. A microelectrophoretic method has been devised for the determination of the basic nuclear protein content of accurately identified homogeneous stages of spermatid differentiation. No significant difference was observed during the first stages of spermiogenesis, i.e., in round spermatids, where a typical histone complement was present. At the beginning of nuclear elongation, two new basic protein fractions appeared and coexisted for some time with typical histones; they replaced somatic histones progressively. Later, during elongation, four proteins of high electrophoretic mobility appeared and gradually replaced the intermediary basic proteins. In elongated spermatids, DNA was found tightly packed by these four proteins: three are arginine- and cysteine-rich (Z1, Z2 and S4), the fourth is arginine-rich (Z3). At first, these fractions are all soluble in 0.25 M HCl but during sperm maturation only one (Z3) remains acid-soluble, the others being extractable only after reducing and alkylating treatments. This modification of solubility of Z1, Z2 and S4 corresponded to the oxidation of cysteine residues to form ---S---S--- crosslinks in chromatin of mature sperm cells. Thus spermiogenesis of the dogfish shows two basic nuclear protein transitions which both occur during nuclear elongation.     

Basic protein changes during the final stages of sperm maturation in the house cricket

Polyacrylamide gel electrophoresis has been used to analyse basic protein changes during the final stages of spermiogenesis in the house cricket. Mature sperm were obtained from the spermathecae of inseminated females. Their basic protein is electrophoretically heterogeneous, with two major and two minor components, all of unusually high mobilities, as expected ofprotamine. No histones are present. Testis also contains basic protein components of high mobilities, although in small amount relative to the histones present. Testis preparations were centrifuged on a density gradient of colloidal silica to separate nuclei of different stages of spermiogenesis from each other, and it was found that very late spermatids contain relatively large amounts of protamine. At least seven different protamine-like components, each with a different mobility, occur during the final maturation stages. The particular components present, and their abundancies, vary during development. The complement first found in spermatids is different from that of a later spermatid; still another complement is found in sperm from the seminal vesicle; and still another in mature sperm. Components which are abundant in spermatids are progressively eliminated, while components which are barely detectable in them gradually increase in abundance to become the major components of the basic protein complement at maturity.     

Novel aspect of perinuclear theca assembly revealed by immunolocalization of non-nuclear somatic histones during bovine spermiogenesis

The perinuclear theca (PT) is an important accessory structure of the sperm head, yet its biogenesis is not well defined. To understand the developmental origins of PT-derived somatic histones during spermiogenesis, we used affinity-purified antibodies against somatic-type histones H3, H2B, H2A, and H4 to probe bovine testicular tissue using three different immunolocalization techniques. While undetectable in elongating spermatid nuclei, immunoperoxidase light microscopy showed all four somatic histones remained associated to the caudal head region of spermatids from steps 11 to 14 of the 14 steps in bovine spermiogenesis. Immunogold electron microscopy confirmed the localization of somatic histones on two nonnuclear structures, namely transient manchette microtubules of step-9 to step-11 spermatids and the developing postacrosomal sheath of step-13 and -14 spermatids. Immunofluorescence demonstrated somatic histone immunoreactivity in the developing postacrosomal sheath, and on anti-beta-tubulin decorated manchette microtubules of step-12 spermatids. Focal antinuclear pore complex labeling on the base of round spermatid nuclei was detected by electron microscopy and immunofluorescence, occurring before the nucleoprotein transition period during spermatid elongation. This indicated that, if nuclear histone export precedes their degradation, this process could only occur in this region, thereby questioning the proposed role of the manchette in nucleocytoplasmic trafficking. Somatic histone immunodetection on the manchette during postacrosomal sheath formation supports a role for the manchette in PT assembly, signifying that some PT components have origins in the distal spermatid cytoplasm. Furthermore, these findings suggest that somatic histones are de novo synthesized in late spermiogenesis for PT assembly.     

Changes in distribution of basic nuclear proteins and chromatin organization during spermiogenesis in the greater bandicoot rat, <Emphasis Type="Italic">Bandicota indica</Emphasis>

Male germ cells of the greater bandicoot rat, Bandicota indica, have recently been categorized into 12 spermiogenic steps based upon the morphological appearance of the acrosome and nucleus and the cell shape. In the present study, we have found that, in the Golgi and cap phases, round spermatid nuclei contain 10-nm to 30-nm chromatin fibers, and that the acrosomal granule forms a huge cap over the anterior pole of nucleus. In the acrosomal phase, many chromatin fibers are approximately 50 nm thick; these then thickened to 70-nm fibers and eventually became 90-nm chromatin cords that are tightly packed together into highly condensed chromatin, except where nuclear vacuoles occur. Immunocytochemistry and immunogold localization with anti-histones, anti-transition protein2, and anti-protamine antibodies suggest that histones remain throughout spermiogenesis, that transition proteins are present from step 7 spermatids and remain until the end of spermiogenesis, and that protamines appear at step 8. Spermatozoa from the cauda epididymidis have been analyzed by acid urea Triton X-100 polyacrylamide gel electrophoresis for basic nuclear proteins. The histones, H2A, H3, H2B, and H4, transitional protein2, and protamine are all present in sperm extracts. These findings suggest that, in these sperm of unusual morphology, both transition proteins and some histones are retained, a finding possibly related to the unusual nuclear form of sperm in this species.     

Migration of centromere proteins in rabbit spermatids.

Human autoantibodies were used to localize centromere proteins by immunoelectron microscopy, immunofluorescence, and confocal microscopy in isolated cells and in cryosections of rabbit testis. A computer-assisted three-dimensional reconstruction of the positions and sizes of fluorescent spots allowed us to follow their movements during the different phases of spermiogenesis. In very young spermatids, the centromeres were distributed within a space separated from both the external nuclear limits and the nuclear core. They moved towards the nuclear center in cap phase spermatids, where they clustered into a few large centromeric masses. In preelongating spermatids, the immunolabeled proteins were dispersed within an equatorial area, where they formed one large mass. In late spermatids, the mass moved towards the posterior part of the nucleus, and, in the spermatozoon, the two basal knobs located at the base of the nuclei were the only strongly immunolabeled structures, while no labeling of the main part of the nucleus was observed. Since the number of centromeres remained close to the number of chromosomes until the cap phase of spermatid differentiation, we hypothesize that the labeling of young spermatids corresponds to centrometric proteins associated with their specific DNA counterparts, while the centromere proteins, possibly detached from their DNA loci, were released from nuclei of old spermatids in the same way as are histones and transition proteins.     

Persistence of protamine precursors in mature sperm nuclei of the mouse

During mouse spermiogenesis, two protamines, mP1 and mP2, are synthesized in replacement of histones. One of them (protamine mP2, 63 residues) appears at first in elongating spermatid nuclei as a pro-protamine of 106 residues (pmP2) with an amino-terminal extension that is progressively excised. The two protamines were previously described as the only proteins associated with DNA in sperm chromatin. This paper shows that the nuclear proteins of mouse spermatozoa are indeed heterogeneous: at least six minor polypeptides in addition to protamines can be identified. The primary structure of four of them has been established. They are intermediate in the maturation of the precursor of protamine mP2 and correspond to polypeptides pmP2/11, pmP2/16, pmP2/20, and pmP2/32, characterized previously in mouse testis. Therefore, these intermediates of proteolysis generated from pmP2 inside spermatid nuclei persist in mature sperm, whereas the largest precursors, pmP2 and pmP2/5, disappear. These findings clearly indicate that limited proteolysis events still occur outside of the testis. © 1995 Wiley-Liss, Inc.     

Histones of spermatogenous cells in the house cricket

Histones were isolated from testis of the house cricket and analyzed by electrophoresis on polyacrylamide gels containing urea and acetic acid. Testes of two different nymphal instars and of adults were examined. The testes contained gonial and meiotic stages in the younger nymphs analyzed, and these same stages plus early spermatids in the older nymphs. At both nymphal instars, testis histone displayed the same five major fractions that are found in somatic nuclei of the cricket; the only unusual feature noted in nymph testis was a high abundance of phosphorylated F1. Adult testis has the same histone fractions as nymph testis and has two new fractions in addition. SDS electrophoresis also shows the presence of two more histones in adult testis than in nymph testis. — The unusual testis histones appear to accumulate during the nuclear elongation stages of spermiogenesis. The occurrence of these stages in adult testis is correlated with the presence of the extra histones. Nuclei of adult testis were separated into fractions enriched for early, mid, and late stages by velocity sedimentation at unit gravity. The unusual histones predominate in the fractions enriched for late spermiogenic stages. Both of the new histones appear to occur in the same stages of spermiogenesis, and display linked accumulation. Eventually they make up at least seventy percent of the histone complement.     

Temporal association of protamine 1 with the inner nuclear membrane protein lamin B receptor during spermiogenesis

During mammalian spermiogenesis, histones are replaced by transition proteins, which are in turn replaced by protamines P1 and P2. P1 protamine contains a short arginine/serine-rich (RS) domain that is highly phosphorylated before being deposited into sperm chromatin and almost completely dephosphorylated during sperm maturation. We now demonstrate that, in elongating spermatids, this phosphorylation is required for the temporal association of P1 protamine with lamin B receptor (LBR), an inner nuclear membrane protein that also possesses a stretch of RS dipeptides at its nucleoplasmic NH(2)-terminal domain. Previous studies have shown that the cellular protein p32 also binds tightly to the unmodified RS domain of LBR. Extending those findings, we now present evidence that p32 prevents phosphorylation of LBR and furthermore that dissociation of this protein precedes P1 protamine association. Our data suggest that docking of protamine 1 to the nuclear envelope is an important intermediate step in spermiogenesis and reveal a novel role for SR protein kinases and p32.     

Isolation and characterization of the spermatid-specific Smrp1 gene encoding a novel manchette protein

The manchette, which is the structure that appears around the nuclei of elongated spermatids, is assumed to be involved in nuclear shaping during spermiogenesis and the transport of various proteins between the nucleus and sperm tail. In this report, we describe the molecular cloning and characterization of a mouse spermatid-specific manchette-related protein 1 (Smrp1) from a spermatid-specific subtracted mouse testis cDNA library. The isolated Smrp1 cDNA clones could be divided into three variants based on sequence analysis. Computer-assisted analysis showed that these variants were splice variants from a single locus of the mouse genome. The three putative proteins consisted of 296, 260, and 175 amino acids, respectively. Although 155 amino acids of the N terminus were common to the three proteins, they were distinguished by their C-terminal regions. Western blot analyses using specific antisera showed that SMRP1 expression was specific to the testes and that only the 261-amino-acid form was translated into protein. Immunohistochemistry revealed that SMRP1 was localized to the cytoplasm of step 9-12 elongated spermatids. The protein appeared in a cap formation that covered the caudal sides of the elongated nuclei. This localization pattern coincided with that of the manchette. SMRP1 may play an important role as a functional protein that co-operates with manchette proteins.     

Proteolysis of somatic type histones in transforming rat spermatid chromatin

Elongated rat spermatid nuclei have been isolated on the basis of their resistance to sonication in 0.32 M sucrose containing 1.5 mM CaCl₂. Chemical analyses indicate that approx. 35% of the DNA in these nuclei is associated with somatic type histones, while the remainder represents sperm histone-DNA complex. In contrast to nuclei of somatic cells, when elongated spermatid nuclei are incubated under appropriate conditions, somatic type histones but not sperm histone are rapidly degraded. Differential extraction of elongated spermatid nuclei with 5 mM HCl and then with various concentrations of NaCl followed by 0.2 M HCl has revealed that they contain two kinds of proteases. The protease in the 5 mM HCl extract is acrosin (EC 4.3.21.10). Rapid degradation of somatic type histones is, however, observable upon incubation of elongated spermatid nuclei which have been treated with 5 mM HCl and are therefore free of acrosin or upon incubation of elongated spermatid chromatin where the majority of acrosin is removed, suggesting that the observed proteolysis of somatic type histone is not due to acrosin. Proteases which may represent the enzymes responsible for the histone degradation are extractable from acrosin-free spermatid nuclei with NaCl (0.9 M) and by subsequent treatment of the salt-extracted nuclei with 0.2 M HCl. The proteases in the NaCl and the 0.2 M HCl extract possess identical properties and appear to be the same enyzyme which may exist in spermatid chromatin in two different forms.     

Molecular cloning and characterization of phosphatidylcholine transfer protein-like protein gene expressed in murine haploid germ cells

We have isolated a cDNA clone specifically expressed in spermiogenesis from a subtracted cDNA library of mouse testis. The cDNA consisted of 1392 nucleotides and had an open reading frame of 873 nucleotides encoding a protein of 291 amino acid residues. Computer-mediated homology search revealed that the nucleotide sequence was unique but the deduced amino acid sequence had similarity to mouse phosphatidylcholine transfer protein (PCTP). We named this newly isolated gene PCTP-like protein. Northern blot analysis revealed a 1.4-kilobase mRNA expressed in the testis, kidney, liver, and intestine with the highest level in the testis. Messenger RNA expression in the testis was detected first on Day 23 in postnatal development and then increased up to adulthood. The protein, having a molecular weight of approximately 40 000, was encoded by the mRNA and was detected at the tail of the elongated spermatids and sperm by immunohistochemical staining.