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.     

Glucocorticoid regulation of two serine hydrolases in rat splenic lymphocytes in vitro

A quantitative assay employing binding of [³H]diisopropylfluorophosphate ([³H]DFP) and SDS-polyacrylamide gel electrophoresis was used to measure serine hydrolases in cell-free extracts from rat splenic lymphocytes. After labeling with [³H]DFP at pH 7, six major serine hydrolases are detected on 10% gels, having molecular weights of 78, 55, 34, 30, 28 and 17 (· 10^?3). When labeled at pH 4, only four activities are measured, with M_r or 79, 55, 33 and 17 (· 10^?3). Incubation of splenic lymphocytes for 8 h in vitro with 1 μM dexamethasone followed by [³H]DFP labeling at pH 7 produces a 91% increase in the 17000 [³H]DFP. Hormone treatment for 8 h with subsequent labeling at pH 4 results in a 15% increase in the largest (78000) species, as well as 73% increase in the 17000 enzyme, compared with lysates from cells incubated without steroid. These effects are not observed after only 4 h of glucocorticoid exposure. Dexamethasone treatment for 8 h does not produce a decrease in any of these serine hydrolases, nor is there an apparent induction of new enzymes (i.e., having a molecular weight different from the preexisting species). Studies examining the effect of protease inhibitors on the [³H]DFP capacity of these proteins, show that the 17000 enzyme is sensitive to the protease inhibitor, pepstatin A, as well as the sulfhydryl reagents dithiothreitol and N-ethylmaleimide. These result suggest that this dexamethasone-responsive enzyme is a protease which requires a free thiol group for optimal activity. These findings are discussed with regard to the mechanism of glucocorticoid action in lymphocytes.     

Identification of a nuclear localization signal in activin/inhibin betaA subunit; intranuclear betaA in rat spermatogenic cells

Activin is a dimeric glycoprotein hormone that was initially characterized by its ability to stimulate pituitary FSH secretion and was subsequently recognized as a growth factor with diverse biological functions in a large variety of tissues. In the testis, activin has been implicated in the auto/paracrine regulation of spermatogenesis through its cognate cell membrane receptors on Sertoli and germ cells. In this study we provide evidence for intranuclear activin/inhibin betaA subunit and show its distribution in the rat seminiferous epithelium. We have shown by transient expression in HeLa cells of beta-galactosidase fusion proteins that the betaA subunit precursor contains a functional nuclear localization signal within the lysine-rich sequence corresponding to amino acids 231-244. In all stages of the rat seminiferous epithelial cycle, an intense immunohistochemical staining of nuclear betaA was demonstrated in intermediate or type B spermatogonia or primary spermatocytes in their initial stages of the first meiotic prophase, as well as in pachytene spermatocytes and elongating spermatids primarily in stages IX-XII. In some pachytene spermatocytes, the pattern of betaA immunoreactivity was consistent with the characteristic distribution of pachytene chromosomes. In the nuclei of round spermatids, betaA immunoreactivity was less intense, and in late spermatids it was localized in the residual cytoplasm, suggesting disposal of betaA before spermatozoal maturation. Immunoblot analysis of a protein extract from isolated testicular nuclei revealed a nuclear betaA species with a molecular mass of approximately 24 kDa, which is more than 1.5 times that of the mature activin betaA subunit present in activin dimers. These results suggest that activin/inhibin betaA may elicit its biological functions through two parallel signal transduction pathways, one involving the dimeric molecule and cell surface receptors and the other an alternately processed betaA sequence acting directly within the nucleus. According to our immunohistochemical data, betaA may play a significant role in the regulation of nuclear functions during meiosis and spermiogenesis.     

Daily sperm production of zebus (Bosindicus) estimated by quantitative histology of the testis

Microscopic parameters were studied quantitatively in testes from six Nelore zebus ($\text{Bos}$$\text{indicus}$), aged 4 to 6 years, with normal spermatogenesis, which were kept at sexual rest. Ratios of germ cell nuclei, counted in cross sections of seminiferous tubules, indicated that cellular losses in the spermatogenic process of the zebu are higher than those observed in taurines. Daily sperm production, estimated from the number of round spermatids in stage 1 of the cycle of the seminiferous epithelium and the duration of this cycle, was (mean ± SD) 12.2 ± 0.9 × 10⁶ spermatids/gram of testis parenchyma/day and 2.6 ± 0.5 × 10⁹ spermatids/testis/day. These values are smaller than those of taurines.     

Immunohistochemical localization of the calcium/calmodulin-dependent protein phosphatase,calcineurin, in the mouse testis: its unique accumulation in spermatid nuclei

Immunohistochemical localization of a calmodulin-dependent protein phosphatase, calcineurin, was studied in the mouse testis in relation to previous observations showing that calmodulin is unusually rich in spermatogenic stages from mid-pachytene spermatocytes to elongating spermatids. The antibodies raised against calcineurin from scallop testis reacted with subunit B, but not subunit A, of calcineurin isoforms from mouse brain and testis. Indirect immunofluorescence using these antibodies on the mouse testis revealed positive reactions only in the nuclei of round or elongating spermatids: calcineurin started to accumulate in nuclei from the acrosomal cap phase, peaked at the initial stage of nuclear elongation, and decreased thereafter. There was almost no signal in the cytoplasm; spermatogenic cells at other stages, including spermatogonia, spermatocytes, mature sperm, and other somatic cells in the seminiferous tubules were totally negative. Immuno-electron microscopy gave the same result, on the basis of measuring the density of immunogold particles. These results suggest a role for calcineurin in remodeling of the nuclear chromatin in metamorphosing spermatids.     

Transient DNA strand breaks during mouse and human spermiogenesis new insights in stage specificity and link to chromatin remodeling

In the course of mammalian spermiogenesis, a unique chromatin remodeling process takes place within elongating and condensing spermatid nuclei. The histone-to-protamine exchange results in efficient packaging and increased stability of the paternal genome. Although not fully understood, this change in chromatin architecture must require a global but transient appearance of endogenous DNA strand breaks because most of the DNA supercoiling is eliminated in the mature sperm. To establish the extent of DNA strand breakage and the stage specificity at which these breaks are created and repaired, we performed a sensitive terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay to detect in situ DNA strand breaks on both mice and human testis cross sections. In the mouse, we established that DNA strand breaks are indeed detected in the whole population of elongating spermatids between stages IX and XI of the seminiferous epithelium cycle perfectly coincident with the chromatin remodeling as revealed by histone H4 hyperacetylation. Similarly, TUNEL analyses performed on human testis sections revealed an elevated and global increase in the levels of DNA strand breaks present in nuclei of round-shaped spermatids also coincident with chromatin remodeling. The demonstration of the global character of the transient DNA strand breaks in mammalian spermiogenesis suggests that deleterious consequences on genetic integrity of the male gamete may arise from any disturbance in the process. In addition, this investigation may shed some light on the origin of the low success rate that has been encountered so far with intracytoplasmic injection procedures making use of round spermatids in humans.     

Changes in basic chromosomal proteins during spermatogenesis in the mature rat.

Nuclei of the seminiferous epithelial cells of rat testis were filtered through glass wool to remove sperm heads, flagellae and late-stage spermatids and then centrifuged through sucrose gradients to yield three fractions. The cellular origins of the predominant nuclei in these fractions were identified through the kinetics of labeling with [³H]thymidine. The relative amounts of the different histone fractions changed during the various stages of spermatogenesis in an interesting and systematic manner. For example, the ratio of the trailing (acetylated) to the leading member of the histone F2a1 doublet was greater in spermatid nuclei than in nuclei of a fraction enriched in primary spermatocytes. Similarly, the ratio $\text{X}{}_1\text{F}\text{1}$ was greatest in spermatid nuclei. On the other hand, the ratio $\text{X}{}_3\text{F}\text{2b}$ was greater in the nuclei of pachytene-diplotene primary spermatocytes than in the fraction enriched in nuclei of spermatogonia and preloptotene primary spermatocytes.A basic protein fraction with some of the properties of a protamine was extracted from rat sperm heads and from the nuclei of spermatids. This protein fraction has high contents of arginine and cysteine (after reduction), and it appears to be identical with the protamine described by Kistler et al. In addition, a new protamine was isolated from rat sperm heads which has high arginine content but appears to be devoid of lysine and cyst(e)ine. Two other basic protein fractions with high electrophoretic mobilities were extracted with acid from the nuclei of testicular seminiferous epithetial cells without prior reduction. One of these proteins may be identical with the testis-specific protein of Kistler et al.     

Expression of the neurotransmitter-synthesizing enzyme glutamic acid decarboxylase in male germ cells.

The gene encoding glutamic acid decarboxylase (GAD), the key enzyme in the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid, is shown to be expressed in the testis of several different species. Nucleotide sequence analysis of a cDNA clone isolated from the human testis confirmed the presence of GAD mRNA in the testis. The major GAD mRNA in the testis was 2.5 kilobases. Smaller amounts of a 3.7-kilobase mRNA with the same size as GAD mRNA in the brain was also detected in the testis. In situ hybridization using a GAD-specific probe revealed GAD mRNA expressing spermatocytes and spermatids located in the middle part of rat seminiferous tubules. Studies on the ontogeny of GAD mRNA expression showed low levels of GAD mRNA in testes of prepubertal rats, with increasing levels as sexual maturation is reached, compatible with GAD mRNA expression in germ cells. In agreement with this, fractionation of cells from the rat seminiferous epithelium followed by Northern (RNA) blot analysis showed the highest levels of GAD mRNA associated with spermatocytes and spermatids. Evidence for the presence of GAD protein in the rat testis was obtained from the demonstration of GAD-like immunoreactivity in seminiferous tubules, predominantly at a position where spermatids and spermatozoa are found. Furthermore, GAD-like immunoreactivity was seen in the midpiece of ejaculated human spermatozoa, the part that is responsible for generating energy for spermatozoan motility.     

Immunohistochemical study of nuclear changes associated with male germ cell death and spermiogenesis

In a previous study on the effects of gestational and lactational exposure of para-nonylphenol on male rats, we noted in both induced and uninduced rats, that variations in cleaved caspase-3 immunostaining patterns were associated with distinct nuclear alterations in mainly basally located germ cells (spermatogonia and preleptotene spermatocytes). These were re-analysed and compared with cleaved caspase-3-labeled germ cells in the aging human and the spermatogenically active catfish testis. In the rat testes, cytoplasmic immunostaining was progressively associated with lateral compression of the nucleus, its break up into large pieces which can contain immunostained marginated chromatin masses. The pale remnants of the nucleus continued to shrink in size concomitant with the appearance of blue-purplish stained regions in the cytoplasm similar in color to the condensed chromatin in spermatids, a condition which was TUNEL-negative. These large clumps of chromatin also eventually disappeared, giving rise to cells resembling cytoplasmic ghosts, a condition which was TUNEL-positive. By contrast, the immunolabeled nuclei of human and catfish germ cells condensed into a single mass, after which they lost immunoreactivity. To exclude the possibility that these observations could reflect alterations in Sertoli nuclei, rat testicular sections were probed with a mouse anti-human GATA-4 monoclonal (MHM) antibody. The MHM was, however, the second of two GATA-4 antibodies tested, with a goat anti-mouse polyclonal (GMP) initially used to label the rat Sertoli nuclei. GMP unexpectedly, but distinctly labeled the complete development of the acrosome in the rat testis, a fortuitous finding with utility for staging of the seminiferous epithelium.     

Nuclear protein transitions in cuttle-fish spermiogenesis: Immunocytochemical localization of a protein specific for the spermatid stage

The changes in basic nuclear proteins throughout cuttle-fish spermiogenesis were investigated both by immunocytochemical procedures and by isolation of late spermatid nuclei (by virtue of their resistance to sonication). Antibodies were raised in rabbits to a protein, named protein T, isolated from testis chromatin. The anti-protein T immune serum was found to recognize protein T and not histones from the testis. Immunoperoxidase staining of sections or of smears of testis with anti-protein T antibodies showed that protein T appears in the nuclei of round spermatids, is abundant in elongating spermatid nuclei, but cannot be detected in elongated spermatids. Nuclei from these elongated spermatids were isolated by sonication treatment of testis cells. A protein, named protein Sp, with the characteristic mobility of a protamine, was isolated from elongated spermatid nuclei. This protein has the same mobility as the protamine present in mature spermatozoa. Taken together, the results indicate that in cuttle-fish, nuclear protein transitions involve the replacement of histones by a spermatid-specific protein (protein T), which is replaced at the end of elongation of the nucleus by a protamine (protein Sp). Thus, spermiogenesis of the cuttle-fish (and perhaps of other cephalopods), shows two basic nuclear protein transitions, which are similar to the transitions observed in higher vertebrates such as mammals.     

Increased accessibility of the N-terminus of testis-specific histone TH2B to antibodies in elongating spermatids

Changes in chromatin structure during spermatogenesis were investigated using a monoclonal antibody that immunoreacts with the N-terminus of the testis-specific histone TH2B. This monoclonal antibody, which had been raised against rat tyrosine hydroxylase (TH), cross-reacted with TH2B because of sequence homology at the N-termini of TH and TH2B. The epitope was localized to the N-terminus of TH2B as trypsin-digested chromatin which lacked the N-terminal tail did not react with anti-TH and preincubating anti-TH with a synthetic peptide made from the homologous sequence between TH2B and TH inhibited its binding to TH and TH2B. In histological sections of rat testis, the primary spermatocytes and round spermatids immunoreacted weakly, whereas elongating spermatids at steps 10–12 immunoreacted intensely with anti-TH. Increased staining of elongating spermatids was also observed in mouse and hamster by immunohistochemistry. However, immunoblotting proteins extracted from separated rat testis cells showed no increase in the TH2B content of these late steps of spermatids. The apparent increase in the immunohistochemical staining corresponds to increased accessibility of the epitope in the elongating spermatids. This indicated that the N-terminus of TH2B is less tightly bound to DNA or to other proteins at this time in preparation for the removal of TH2B and other histones. © 1995 wiley-Liss, Inc.     

Intron retention generates a novel isoform of CEACAM6 that may act as an adhesion molecule in the ectoplasmic specialization structures between spermatids and sertoli cells in rat testis

By differential display technique followed by RT-PCR and DNA sequence analyses, we isolated carcinoembryonic antigen-related cell adhesion molecule 6 (Ceacam6) and its novel spliced variant Ceacam6-Long (Ceacam6-L) from rat testis. Ceacam6-L mRNA was generated by retention of 67 nucleotide-length third intron in Ceacam6 gene. Ceacam6-L is a member of an immunoglobulin superfamily and encodes a protein of 50 kDa with a signal sequence at the N-terminus, one immunoglobulin (Ig)-like domain, three IgCAM domains, a transmembrane region, and a short intracellular region. Expression analyses by RT-PCR and Northern blot showed that Ceacam6-L was exclusively expressed in rat testis and first detectable at 5 wk during postnatal development of testis. We performed immunoblot analyses and immunohistochemistry using the anti-CEACAM6-L antibody. Confocal laser scanning microscopy revealed that CEACAM6-L was not present at blood-testis barrier junctions between Sertoli cells but localized at the interface between Sertoli cells and germ cells, possibly to work as an adhesion molecule in the apical compartment of the seminiferous epithelium. At stages VII-VIII, at which all of the elongated spermatids migrated to the luminal surface of the seminiferous tubules, CEACAM6-L was found to locate at the concave side of elongated spermatid heads, following the curvature of their sickle-shaped nuclei, suggesting that CEACAM6-L might be involved in the anchoring of spermatids to Sertoli cells and spermiation. We concluded that CEACAM6-L might be a novel adhesion molecule constructing the apical ectoplasmic specialization in testis.     

Isolation of rat testis histone TH2B-x, and interaction of TH2B-x antiserum with histones and mononucleosomes

A method is reported for the isolation of histone TH2B-x from rat testis by affinity chromatography on an agarose-p-chloromercurianilino column. This purified TH2B-x was used to raise antibodies in the rabbit, and the antiserum was assayed by an enzyme-linked double-antibody procedure. At low concentration the antiserum cross-reacts with histone H2B and with histones TH1-x + H1 to the extent of 11-14% of the interaction with TH2B-x. Antiserum preincubated in three successive H2B-coated tubes still retains 80-89% of the original anti-TH2B-x activity when assayed subsequently in TH2B-x-coated tubes, but cross-reaction with H2B is practically zero. The anti-TH2B-x antibodies also interact with tubes coated with mononucleosomes isolated from nuclei of seminiferous epithelial cells (SEC) of rat testis, but the interaction with mononucleosomes from rat liver nuclei is almost zero. The data suggest that in nucleosomes some of the antigenic determinants which are unique to TH2B-x are accessible, while those determinants which are common to H2B and TH2B-x are not accessible for interaction with antibodies. Competition by mononucleosomes, both from rat testis SEC and rat liver (to a lesser degree), in solution is detected by the reduction of binding of enzyme-labeled IgG to TH2B-x-coated tubes. However, an attempted competition by histones TH2B-x or H2B in solution resulted in an increase in the binding of the enzyme-labeled IgG to the mononucleosome-coated tubes. The interpretation of this type of competition assay is complicated by possible interaction of added histones with the coating mononucleosomes, followed by binding of antibodies to the histones. This TH2B-x antibody should be useful in studying changes in structure and function of chromatin during spermatogenesis and in the isolation of TH2B-x mRNA.     

Characterization of high mobility group protein levels during spermatogenesis in the rat

The distribution, quantitation, and synthesis of high mobility group (HMG) proteins during spermatogenesis in the rat have been determined. HMG1, -2, -14, and -17 were isolated from rat testes by Bio-Rex 70 chromatography combined with preparative gel electrophoresis. Amino acid analysis revealed that each rat testis HMG protein was similar to its calf thymus analogue. Tryptic peptide maps of somatic and testis HMG2 showed no differences and, therefore, failed to detect an HMG2 variant. Testis levels of HMG proteins, relative to DNA content, were equivalent to other tissues for HMG1 (13 micrograms/mg of DNA), HMG14 (3 micrograms/mg of DNA), and HMG17 (5 micrograms/mg of DNA). The testis was distinguished in that it contained a substantially higher level of HMG2 than any other rat tissue (32 micrograms/mg of DNA). HMG protein levels were determined from purified or enriched populations of testis cells representing the major stages of spermatogenesis; spermatogonia and early primary spermatocytes, pachytene spermatocytes, early spermatids, and late spermatids; and testicular somatic cells. High levels of HMG2 in the testis were due to pachytene spermatocytes and early spermatids (56 +/- 4 and 47 +/- 6 micrograms/mg of DNA, respectively). Mixtures of spermatogonia and early primary spermatocytes showed lower levels of HMG2 (12 +/- 3 micrograms/mg of DNA) similar to proliferating somatic tissues, whereas late spermatids had no detectable HMG proteins. The somatic cells of the testis, including isolated populations of Sertoli and Leydig cells, showed very low levels of HMG2 (2 micrograms/mg of DNA), similar to those in nonproliferating somatic tissues. HMG proteins were synthesized in spermatogonia and primary spermatocytes, but not in spermatids. Rat testis HMG2 exhibited two bands on acid-urea gels. A "slow" form comigrated with somatic cell HMG2, while the other "fast" band migrated ahead of the somatic form and appeared to be testis-specific. The "fast" form of HMG2 accounted for the large increase of HMG2 levels in rat testes. These results show that the very high level of HMG2 in testis is not associated with proliferative activity as previously hypothesized.     

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.     

Molar proportions and relative rates of synthesis of histones in rat testis

The molar proportions and relative rates of synthesis of histones in normal and hypophysectomized rat testis seminiferous epithelial cells were determined. After hypophysectomy the molar proportions of histones H1, H2B and (H2A + protein A24) in seminiferous epithelial cells of rat testis increased while their corresponding variants TH1-x, TH2B-x and X₂ decreased, but the molar proportions of major-class histones (i.e., sum of subfractions) remained relatively constant and similar to the proportions in somatic cells. The apparent molar proportions of the labeled histones, determined immediately after 2-h periods of [³H]leucine incorporation, were much higher relative to H4 than the proportions of total histones determined by dye binding. The values, however, approached the molar proportions of total histones when rats were killed 11 days after the [³H]leucine injection. Two-dimensional gel electrophoresis confirmed that the high initial molar proportions relative to H4 by [³H]leucine incorporation were not due to the possible contamination by highly-labeled non-histone proteins. The specific activity of histone H4 relative to the specific activity of DNA, determined immediately after 3-h periods of [³H]leucine and [¹⁴C]thymidine incorporations was similar to the value when rats were killed 13 days after the injections. It is proposed that histones of seminiferous epithelial cells are synthesized disproportionally relative to H4 and in excess of the quantities required for polynucleosome assembly. The excess histones are subsequently displaced or degraded slowly.