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.     

Effect of inhibition of DNA synthesis on histone synthesis, turnover, and deposition in the rat testis

In testicular seminiferous epithelial cells (SEC) of normal and hypophysectomized rats, 1-beta-D-arabinofuranosylcytosine and hydroxyurea (at concentrations which inhibited DNA synthesis nearly completely) inhibited histone synthesis only partially, and to a different extent for each histone fraction. In the presence of the inhibitors, the extent of synthesis relative to the corresponding control was TH1-x greater than H1 greater than TH2B-x = X2 = H2A greater than H2B = H3 greater than H4, in which synthesis of the H4 fraction was about 50% of control and that of TH1-x was 90-95% of control. The extent of inhibition of synthesis of each histone fraction was similar after hypophysectomy and, therefore, the changing of the relative populations of heterogeneous cells in the SEC did not influence the relative effects of the inhibitors of DNA synthesis on the synthesis of the various histone fractions. After [3H]leucine injection, the molar proportions of labeled histones relative to H4 decreased markedly between 1.5 h and 6-15 days; this finding indicated that there was rapid removal of histones compared to the H4 fraction during this period. When [14C]thymidine was injected 24 h prior to hydroxyurea treatment and [3H]leucine injection, the ratios of specific activities of histone H4 to DNA did not change significantly over an 11-day period. It appears that newly synthesized histone H4 and other somatic histones are associated with existing DNA in the presence of DNA inhibitors.     

Spermatogonial Multiplication In the Chinese Hamster. Iv. Search For Long Cycling Stem Cells

ABSTRACT In the Chinese hamster, 17 days, i. e. one cycle of the seminiferous epithelium, after two injections of [³H]TdR given 24 hr apart, labelled cells were found among all types of spermatogonia, including stem cells (A_s). These labelled A_s spermato-gonia derive from one or more self-renewing divisions of the stem cells that originally incorporated [³H]TdR. In the steady state, half of the divisions of the A_s will be self-renewing and the other half will give rise to A_pr spermatogonia that will ultimately become spermatozoa. Theoretically, the labelling index (LI) after 17 days will be similar to that after 1 hr, and in this study twice as high as for the 1-hr interval since only one injection was given. However, experimental values only half that of the theoretical LI were found after 17 days. the following causes for the loss of labelled stem cells are discussed: (1) dilution of label because of division; (2) influx of unlabelled components of false pairs (i. e. newborn stem cells that still have to migrate away. mostly during G₁, from their sister cells and are scored as A_pr spermatogonia) between 1 hr and 17 days; (3) the existence of long- and short-cycling stem cells, probably combined with preferential differentiation of the short-cycling elements; (4) selective segregation of DNA at stem cell mitosis; and (5) irradiation death of radiosensitive labelled stem cells. As it is not impossible that factors 1, 2, 4 and 5 together account for the total loss of labelled stem cells, LI results do not provide evidence for the existence of separate classes of short- and long-cycling stem cells. The distributions of the LIs of the A_s, A_pr and A_al spermatogonia over the stages of the epithelial cycle at 17 days are similar to those at 1 hr after injection. Hence the regulatory mechanisms that govern the stimulation and inhibition of proliferation of A_s that give rise to new A_s for the next epithelial cycle are similar to those of the A_s that will divide into A_pr spermatogonia during the same epithelial cycle. Grain counts revealed that more [³H]TdR is incorporated into A_s, A_pr and A_al spermatogonia that are in S phase during epithelial stages X-IV than in stages V-IX.     

Preliminary study of sperm chromatin characteristics of the brachyuran crab Maja brachydactyla. Histones and nucleosome-like structures in decapod crustacean sperm nuclei previously described without SNBPs

An interesting characteristic of decapod crustacean sperm nuclei is that they do not contain highly packaged chromatin. In the present study we re-examine the presence of DNA-interacting proteins in sperm nuclei of the brachyuran Maja brachydactyla. Although previous reports have indicated that, unlike the majority of sperm cells, DNA of decapod sperm is not organized by basic proteins, in this work we show that: (1) histones are present in sperm of M. brachydactyla; (2) histones are associated with sperm DNA; (3) histone H3 appears in lower proportions than the other core histones, while histone H2B appears in higher proportions; and (4) histone H3 in sperm nuclei is acetylated. This work complements a previous study of sperm histones of Cancer pagurus and supports the suggestion that decapod crustacean sperm chromatin deserves further attention.     

Spermatogonial multiplication in the Chinese hamster. IV. Search for long cycling stem cells

In the Chinese hamster, 17 days, i.e. one cycle of the seminiferous epithelium, after two injections of [3H]TdR given 24 hr apart, labelled cells were found among all types of spermatogonia, including stem cells (As). These labelled As spermatogonia derive from one or more self-renewing divisions of the stem cells that originally incorporated [3H]TdR. In the steady state, half of the divisions of the As will be self-renewing and the other half will give rise to Apr spermatogonia that will ultimately become spermatozoa. Theoretically, the labelling index (LI) after 17 days will be similar to that after 1 hr, and in this study twice as high as for the 1-hr interval since only one injection was given. However, experimental values only half that of the theoretical LI were found after 17 days. The following causes for the loss of labelled stem cells are discussed: (1) dilution of label because of division; (2) influx of unlabelled components of false pairs (i.e. newborn stem cells that still have to migrate away, mostly during G1, from their sister cells and are scored as Apr spermatogonia) between 1 hr and 17 days; (3) the existence of long- and short-cycling stem cells, probably combined with preferential differentiation of the short-cycling elements; (4) selective segregation of DNA at stem cell mitosis; and (5) irradiation death of radiosensitive labelled stem cells. As it is not impossible that factors 1, 2, 4 and 5 together account for the total loss of labelled stem cells, LI results do not provide evidence for the existence of separate classes of short- and long-cycling stem cells. The distributions of the LIs of the As, Apr and Aal spermatogonia over the stages of the epithelial cycle at 17 days are similar to those at 1 hr after injection. Hence the regulatory mechanisms that govern the stimulation and inhibition of proliferation of As that give rise to new As for the next epithelial cycle are similar to those of the As that will divide into Apr spermatogonia during the same epithelial cycle. Grain counts revealed that more [3H]TdR is incorporated into As, Apr and Aal spermatogonia that are in S phase during epithelial stages X-IV than in stages V-IX.     

Feedback Regulation of the Proliferation of the Undifferentiated Spermatogonia In the Chinese Hamster By the Differentiating Spermatogonia

In the seminiferous epithelium the differentiating spermatogonia proliferate following a very strict synchronous pattern, and undergo the S phase during parts of particular epithelial stages. the undifferentiated spermatogonia do not divide synchronously and display maximum proliferative activity in stages XI-III. Hence the S-phase-specific cytotoxic agent Ara-C kills different proportions of these two cell types dependent on the epithelial stage. We have studied the effect of several combinations of degrees of cell loss to both compartments on proliferation of the undifferentiated spermatogonia. It was found that when the differentiating spermatogonia are removed, the proliferation of the undifferentiated spermatogonia is not inhibited at epithelial stage III, as seen in controls. However, when the undifferentiated spermatogonia were already arrested in G₁, removal of the differentiating spermatogonia did not evoke proliferation again. When the population of undifferentiated spermatogonia was reduced in an area where the differentiating spermatogonia were left intact, the inhibition of the proliferation of undifferentiated spermatogonia took place around stage III as usual. It is concluded that in the normal adult seminiferous epithelium, the length of the period of active proliferation of the undifferentiated spermatogonia is regulated by negative feedback from the differentiating spermatogonia.     

Alterations in amounts and covalent modifications of low-molecular-weight chromosomal proteins in Chinese hamster ovary cells during polyamine depletion

The effect of polyamine depletion on phosphorylation and ADP-ribosylation of low-M_r chromosomal proteins was studied in intact, mutant Chinese hamster ovary cells (CHO-P22) devoid of ornithine decarboxylase activity. When starved of polyamines for 6 days, severe polyamine deficiency develops and the cells gradually stop growing. The rate of DNA synthesis was retarded to 16% of the control value and to 29% in density-inhibited cells. The synthesis of high-mobility-group (HMG) proteins was decreased by 65% in polyamine-depleted cells and by 40% in density-inhibited cells. The synthesis of core histones was decreased by 40% both in polyamine-depleted and density-inhibited cells. In polyamine-depleted cells the molar ratio of the higher-M_r HMG proteins (HMG 1 + 2) to the lower-M_r HMG proteins (HMG 14 + P) was about one-half of that found in cells grown in the presence of putrescine or in density-inhibited cells. In contrast to HMG proteins, no major differences were found in the content of core histones in these cell populations. In the perchloric acid-soluble fraction of nuclear proteins, ³²P was incorporated mainly into histone H1, HMG P and a protein migrating more slowly than HMG 1 (protein P1). Specific changes in the ³²P-labeling and migration of a number of protein bands, including histone H1, was observed in polyamine-depleted cells as compared to cells grown in the presence of putrescine or to density-inhibited cells. ADP-ribosylation experiments using [³H]adenosine showed a different pattern of label distribution; the higher-M_r HMG proteins from polyamine-depleted cells contained about one-half the amount of label found in the proteins from control cells. The lower-M_r HMG proteins and histone H1 were the preferentially labeled proteins in polyamine-depleted cells. Labeling of core histones with [³²P]orthophosphate or [³H]adenosine did not differ markedly in the two cell populations. The results obtained using intact polyamine auxotrophic cells indicated that polyamine depletion is connected with more severe alterations in amounts and covalent modifications (phosphorylation and ADP-ribosylation) of HMG chromosomal proteins and histone H1 than core histones.     

Effects of age and hypophysectomy upon relative proportions of various histones in rat testis.

Various histone fractions of seminiferous epithelial cells of the testis of rats change in relative proportions during development and maturation of the testis with increasing age after birth, and these changes in the histones occur in the reverse direction in testis of mature rats during involution of the testis resulting from hypophysectomy. The changes are particularly striking in histone sub-fractions, designated X₁, X₂, and X₃, which are especially characteristic of testis and may be involved in meiosis.     

THE SPERMATOGONIAL STEM CELL POPULATION IN ADULT RATS

Radioautographed whole mounted seminiferous tubules from adult rat testes were used to analyse undifferentiated type A spermatogonia at various intervals up to 81 hr following a single injection of ³H-TdR. the data obtained led to the identification of the spermatogonial stem cell and to the formulation of a new model for spermatogonial renewal and differentiation. Undifferentiated type A cells were morphologically alike, but were topographically classified as (1) isolated or (2) paired and aligned. Although labeled isolated A cells were scattered over most stages of the seminiferous epithelium, their proliferative activity varied with the stage; their labeling index was 20-30% in stages I and II, but less than 1% in stages VII and VIII. By tracing the labeled divisions of isolated A spermatogonia in time, it was seen that some daughter cells became separated from one another to form two new isolated cells, while others remained together as paired A spermatogonia. Analysis of two successive waves of labeled mitoses revealed that most paired A spermatogonia continued to proliferate forming four aligned A cells, many of which divided again to produce a chain of eight and so on. the greatest incidence of labeling among paired and aligned A spermatogonia occurred in stages XIII-III. In stage I, where the labeling index was 50%, the calculated proliferative fraction was 1 for these spermatogonia. Between stages II and V, they began to leave mitotic cycle, and during stage V this entire cohort morphologically transformed into A₁ spermatogonia. Labeled metaphase curves for undifferentiated A spermatogonia were distinct from any of the curves previously constructed for the six classes of differentiating spermatogonia, especially because of particularly long S and G₂ phases in the former. the cell cycle time of paired and aligned A cells was 55 hr, compared to an average of 42 hr for differentiating types A₂ to B.     

A quantitative study of spermatogonial multiplication and stem cell renewal in the C3H/101 F1 hybrid mouse

In whole mounts of seminiferous tubules of C3H/101 F₁ hybrid mice, spermatogonia were counted in various stages of the epithelial cycle. Furthermore, the total number of Sertoli cells per testis was estimated using the disector method. Subsequently, estimates were made of the total numbers of the different spermatogonial cell populations per testis.

The results of the cell counts indicate that the undifferentiated spermatogonia are actively proliferating from stage XI until stage IV. Three divisions of the undifferentiated spermatogonia are needed to obtain the number of A₁ plus undifferentiated spermatogonia produced each epithelial cycle. Around stage VIII almost two-thirds of the A_pr and all of the A_al spermatogonia differentiate into A₁ spermatogonia. It was estimated that there are 2.5 × 10⁶ differentiating spermatogonia and 3.3 × 10⁵ undifferentiated spermatogonia per testis. There are about 35,000 stem cells per testis, constituting about 0.03% of all germ cells in the testis. It is concluded that the undifferentiated spermatogonia, including the stem cells, actively proliferate during about 50% of the epithelial cycle.     

Histone synthesis in early amphibian development: histone and DNA syntheses are not co-ordinated

The synthesis of basic proteins has been studied in the oocytes, eggs and embryos of the South African clawed frog, Xenopus laevis. A group of newly synthesized proteins has been identified as histones by the following criteria: solubility properties; incorporation of [³H]lysine and [³H]arginine in the correct proportions, but lack of incorporation of [³H]tryptophan; co-cleotrophoresis with marker histones in various types of polyacrylamide gels, including a type run in two dimensions; peptide analysis of the arginine-rich fraction, F2A1. The four main histone fractions other than F1 were found to be synthesized at all stages of development. F1 histone synthesis was first detected at the late blastula stage.Rates of histone synthesis were estimated for the different stages of development and it was concluded that histone synthesis was not co-ordinated with DNA synthesis either temporally or quantitatively. Histone synthesis was unusual in the following major respects: histones were synthesized in oocytes, and yet in these cells DNA replication had not occurred for several months; histones were synthesized in activated or fertilized eggs at a rate far in excess (about 500 times) of the immediate requirements. We suggest that in order to provide enough histones for the late blastula embryo a store of histone is accumulated during the early cleavage stages and possibly during oogenesis.     

Acetylation of histones of rat testis.

When [1-¹⁴C]acetate was injected into rats intratesticularly in the presence of cycloheximide to inhibit protein synthesis, the label was incorporated into histone fractions F2a1 and F3 and into non-histone chromosomal proteins of each of the following stages of spermatogenesis: spermatogonia-preleptotene spermatocytes, leptotene-zygotene-pachytene-diplotene primary spermatocytes, and spermatids. Acetylation of histones was particularly active in the spermatid stages. There was no significant incorporation of acetate into the lysine-rich histone fractions F1 and X₁.In early periods of in vivo incorporation of [³H]amino acids into histones the acetylated histone F2a1 fractions had higher specific activities than the main band of F2a1, but with the passage of time the label moved into the principal band to the extent that specific activities in the acetylated and principal bands were approximately equal at 6 days. However, at 24–36 days the specific activities were again higher in the acetylated bands than in the principal band of F2a1. These data support the conclusions of Candido, Louie, and Dixon, from experiments with trout testis, that acetylation of histone F2a1 may be important in the process of combination of this protein with DNA in chromatin at the spermatogonia-primary spermatocyte stage and also in the subsequent removal of this histone for replacement by protamines at the spermatid stage.[³H]Amino acids were incorporated into histone fractions X₁ and F1 at approximately equal rates, and there was no evidence that one of these fractions was a precursor of the other.Chromatin of the seminiferous epithelial cells of rat testis has a firmly bound acetylase which catalyzes the in vitro acetylation of histones F3 and F2a1 by acetyl CoA.     

Feedback regulation of the proliferation of the undifferentiated spermatogonia in the Chinese hamster by the differentiating spermatogonia

In the seminiferous epithelium the differentiating spermatogonia proliferate following a very strict synchronous pattern, and undergo the S phase during parts of particular epithelial stages. The undifferentiated spermatogonia do not divide synchronously and display maximum proliferative activity in stages XI-III. Hence the S-phase-specific cytotoxic agent Ara-C kills different proportions of these two cell types dependent on the epithelial stage. We have studied the effect of several combinations of degrees of cell loss to both compartments on proliferation of the undifferentiated spermatogonia. It was found that when the differentiating spermatogonia are removed, the proliferation of the undifferentiated spermatogonia is not inhibited at epithelial stage III, as seen in controls. However, when the undifferentiated spermatogonia were already arrested in G1, removal of the differentiating spermatogonia did not evoke proliferation again. When the population of undifferentiated spermatogonia was reduced in an area where the differentiating spermatogonia were left intact, the inhibition of the proliferation of undifferentiated spermatogonia took place around stage III as usual. It is concluded that in the normal adult seminiferous epithelium, the length of the period of active proliferation of the undifferentiated spermatogonia is regulated by negative feedback from the differentiating spermatogonia.     

Renewal and proliferation of spermatogonia during spermatogenesis in the Japanese quail,Coturnix coturnix japonica

Summary Four different types of spermatogonia were identified in the seminiferous tubules of the Japanese quail: a dark type A (A_d), 2 pale A type (A_p1 and A_p2), and a type B. A model is proposed describing the process of spermatogonial development in the quail. The A_d spermatogonia are considered to be the stem cells. Each divides to produce a new A_d spermatogonium and a A_p1 spermatogonium during Stage IX of the cycle of the seminiferous epithelium. An A_p1 spermatogonium produces two A_p2 spermatogonia during Stage II of the cycle, A_p2 spermatogonia produce four type B spermatogonia during Stage VI of the cycle, and type B spermatogonia produce eight primary spermatocytes during Stage III of the cycle. Consequently, 32 spermatids can result from each division of an A_d spermatogonium. Spermatogonial development in the quail differs from the process described in mammals in that there are fewer mitotic divisions and they are all synchronized with the cycle of the seminiferous epithelium. It is suggested that the fewer mitotic divisions explain why a smaller area of the seminiferous tubule is occupied by a cellular association in the quail than in mammals like the rat, ram and bull. The duration of spermatogenesis from the division of the A_d spermatogonia to sperm release from the seminiferous epithelium was estimated to be 12.77 days.     

Regulation of proliferation and differentiation in spermatogonial stem cells: the role of c-kit and its ligand SCF

To study self-renewal and differentiation of spermatogonial stem cells, we have transplanted undifferentiated testicular germ cells of the GFP transgenic mice into seminiferous tubules of mutant mice with male sterility, such as those dysfunctioned at Steel (Sl) locus encoding the c-kit ligand or Dominant white spotting (W) locus encoding the receptor c-kit. In the seminiferous tubules of Sl/Sl(d) or Sl(17H)/Sl(17H) mice, transplanted donor germ cells proliferated and formed colonies of undifferentiated c-kit (-) spermatogonia, but were unable to differentiate further. However, these undifferentiated but proliferating spermatogonia, retransplanted into Sl (+) seminiferous tubules of W mutant, resumed differentiation, indicating that the transplanted donor germ cells contained spermatogonial stem cells and that stimulation of c-kit receptor by its ligand was necessary for maintenance of differentiated type A spermatogonia but not for proliferation of undifferentiated type A spermatogonia. Furthermore, we have demonstrated that their transplantation efficiency in the seminiferous tubules of Sl(17H)/Sl(17H) mice depended upon the stem cell niche on the basement membrane of the recipient seminiferous tubules and was increased by elimination of the endogenous spermatogonia of mutant mice from the niche by treating them with busulfan.     

Renewal of spermatogonia in the monkey (Macaca fascicularis)

Populations of different types of spermatogonia and their mitotic activity were analyzed in the monkey Macaca fascicularis: 3 adults aged 5-6 yr and 3 young aged 2-3 mo. Two young and two adult monkeys received injections of 3H-thymidine for radioautographic study of the relationships between Type A spermatogonia: dark Type A (Ad), pale Type A (Ap) and transition Type A (At). In the adult the number of Ad and At spermatogonia did not change significantly throughout the seminiferous epithelium cycle. The number of Ap spermatogonia doubled at Stage VII, and half divided at Stage IX to give rise to B1 spermatogonia. The durations of the seminiferous epithelium cycle and spermatogenesis were estimated as 10.5 days and 42 days respectively. In the young and adult monkeys, some Ap spermatogonia and a lesser number of At spermatogonia were labeled one h after injection of precursor. At longer intervals after injection, the number of labeled At spermatogonia increased significantly, and some Ad as well as Ap spermatogonia were also labeled. These results indicate that Ap spermatogonia are renewal stem cells, and Ad spermatogonia are reserve stem cells. The differences in labeling after isotope exposure suggest that Ap cells may give rise successively to At and Ad cells.     

Regulated hyperacetylation of core histones during mouse spermatogenesis: involvement of histone deacetylases

Here we report a detailed analysis of waves of histone acetylation that occurs throughout spermatogenesis in mouse. Our data showed that spermatogonia and preleptotene spermatocytes contained acetylated core histones H2A, H2B and H4, whereas no acetylated histones were observed throughout meiosis in leptotene or pachytene spermatocytes. Histones remained unacetylated in most round spermatids. Acetylated forms of H2A and H2B, H3 and H4 reappeared in step 9 to 11 elongating spermatids, and disappeared later in condensing spermatids. The spatial distribution pattern of acetylated H4 within the spermatids nuclei, analyzed in 3D by immunofluorescence combined with confocal microscopy, showed a spatial sequence of events tightly associated with chromatin condensation. In order to gain an insight into mechanisms controlling histone hyperacetylation during spermiogenesis, we treated spermatogenic cells with a histone deacetylase inhibitor, trichostatin A (TSA), which showed a spectacular increase of histone acetylation in round spermatids. This observation suggests that deacetylases are responsible for maintaining a deacetylated state of histones in these cells. TSA treatment could not induce histone acetylation in condensing spermatids, suggesting that acetylated core histones are replaced by transition proteins without being previously deacetylated. Moreover, our data showed a dramatic decrease in histone deacetylases in condensing spermatids. Therefore, the regulation of histone deacetylase activity/concentration appears to play a major role in controling histone hyperacetylation and probably histone replacement during spermiogenesis.     

Arrest of spermatogonial differentiation in jsd/jsd, Sl17H/Sl17H, and cryptorchid mice.

The nature of the spermatogenic arrest in cryptorchid C57Bl mice and in jsd/jsd and Sl17H/Sl17H mutant mice was identified by studying whole mounts of seminiferous tubules. In all three types of mice, virtually only A spermatogonia were found, topographically arranged in clones of 1 to 16 (rarely more) cells. These clonal sizes are typical for undifferentiated spermatogonia. The proportion of these cells lying in chains of more than 2 cells (50-70%) was comparable to that seen in epithelial stages VII-VIII in the normal epithelium. It is concluded that in all three types of mice, spermatogenesis is arrested at the point where the undifferentiated A spermatogonia, specifically A(al) spermatogonia, differentiate into the first generation of the differentiating-type spermatogonia, the A1 spermatogonia. The remaining A spermatogonia were proliferating, but no accumulation of spermatogonia was present, as spermatogonial apoptosis also took place. Spermatogonial clones of all sizes were seen to undergo apoptosis, but there were relatively many large apoptotic clones, indicating that the clones became more vulnerable when they became larger. In contrast to what is seen in the normal epithelium, odd-numbered clones, not composed of 2(n) cells, were present, as well as clumps of 2 or more spermatogonial nuclei in the same cytoplasm, in all three types of mice. This indicates a lack of integrity of spermatogonial clones, also observed in other situations with a relative paucity of cells on the basal membrane. It is concluded that the differentiation of the undifferentiated spermatogonia, affected in all three types of mice as well as in vitamin A-deficient animals, is a rather vulnerable point in the spermatogenic developmental pathway.