Late Spermatid Stage Research Articles

Effects of ethylene glycol monomethyl ether on various parameters of testicular function in the F344 rat.

These studies were designed to define the target cell for ethylene glycol monomethyl ether (EGME) action in the rodent testis, and to compare fluid production and protein secretory patterns in the testes of treated and control animals. Adult F344 rats were treated with 150 mg/kg/day, 5 day/week, and sacrificed 1, 2, 4, 7 or 10 days after the first dose. In the testes of treated animals, the most sensitive cells were the premeiotic and meiotic spermatocytes. As length of treatment continued, the younger transitional spermatocytes, and more pachytene spermatocytes, were affected. Early and late stage spermatids did not appear affected, nor was the visual appearance of spermatogonia changed by exposure to EGME. Fluid production, androgen-binding protein secretion and electrophoreograms of proteins found in the ligated rete testis were also unchanged by EGME, showing that these indices of Sertoli cell function were not affected by EGME. The histologic and biochemical data suggest that both early and late spermatocytes are targets for EGME in the testis, and that the Sertoli cells are relatively unaffected.

Development of agranular reticulum in Sertoli cells of the testis of the dogfish Squalus acanthias during spermatogenesis.

Biochemical analyses of Squalus testis indicates that key enzymes involved with androgen production increase progressively from immature regions containing spermatogonia to mature regions in the late spermatid stage of maturation (Canick et al., 1983). In an effort to identify cells possessing the cytological characteristics of steroid production and to determine the structural correlates of the observed functional changes, we have carried out an electron microscopic study of Squalus testis. This report demonstrates that Sertoli cells contain a well-developed agranular reticulum, mitochondria with tubulovesicular cristae, and numerous lipid droplets. Moreover, as germ cells mature, there is an increase in abundance of agranular reticulum in the adjacent Sertoli cells. By the time of spermatid elongation, this has reached dramatic proportions and fills the Sertoli cell as a mass of tubules. These results lead us to conclude that the Sertoli cell is responsible for secretion of the increasing amounts of androgen during the spermatogenetic cycle in Squalus.

Mutagenic activity of the pyrrolizidine alkaloids seneciphylline and senkirkine in drosophila and their transfer into rat milk

Seneciphylline and senkirkine, two pyrrolizidine alkaloids that occur in animal feeds and medicinal herbs, respectively, have been tested for their ability to produce sex-linked recessive lethals in males of Drosophila melanogaster using the Basc test (3-day feeding method). Seneciphylline was found to be mutagenic at concentrations of 10 −5, 10 −4 and 10 −3 m, which produced 3.8% sex-linked recessive lethals (983 chromosomes tested), 9.0% (708) and 15.3% (327), respectively. Senkirkine (10 −5 m) produced 4.4% sex-linked recessive lethals (2541 chromosomes tested) against 0.17% (9081) in controls. Brood pattern analysis with senkirkine showed maximum sensitivity in the late spermatid stage of spermatogenesis, which agrees with evidence that pyrrolizidine alkaloids act as indirect mutagens. Flies fed with milk from lactating rats given an oral dose of 25 mg seneciphylline/kg showed 1.2% sex-linked recessive lethals (1477 chromosomes tested), against 0.3% (1533) in controls.

Localization of energy metabolism-related oxidoreductases in the testis.

It is well known that mammalian spermatozoa utilize fructose as an energy source, and glycerol can also be metabolized for this purpose. Further, oxidoreductases related to energy metabolism, such as sorbitol dehydrogenase and α-glycerophosphate dehydrogenase, are contained in the testis. These enzymes contribute fructose synthesis and glycerol metabolism, respectively.In this paper, the precise localization of these energy metabolism-related oxidoreductases was studied by electron microscopy. The results indicated that sorbitol dehydrogenase located in the cytoplasm of Sertoli cells adjacent to the late stage spermatids. On the other hand, α-glycerophosphate dehydrogenase was recognized in the mitochondria of late stage spermatids and spermatozoa. Therefore, fructose is presumably synthesized in the cytoplasm of the Sertoli cells, and glycerol is metabolized at the mitochondria of the sperm.

SPERMIOGENESIS IN BARNACLES WITH SPECIAL REFERENCE TO ORGANIZATION OF THE ACCESSORY BODY.

Ultrastructural changes during spermiogenesis in the barnacles, Balanus amphitrite albicostatus, Balanus tintinnabulum rosa, Balanus trigonus and Tetraclita squamosa japonica, and organization of the sperm with special reference to the accessory body were studied. The Golgi complex organizes both the acrosome and the accessory body at different stages during spermiogenesis; the former is formed at the mid-spermatid stage and the latter is formed at the late spermatid stage. The arrangement of the components in the mature filiform sperm is quite unique, with the acrosome, the basal body just behind the acrosome, the axial filament parallel to a long nucleus, and a slender long mitochondrion behind the nucleus. The sperm in the anterior and posterior half of the ejaculatory duct differ from each other in form in that the sperm in the anterior duct are not equipped with the accessory body and the sperm in the posterior duct are. The accessory body can be artificially broken down by some treatments (1 M urea, alkaline sea water: pH 9.0-9.7, low ionic concentration of sea water). The loss of the accessory body from the sperm is assumed to be related to the ferti-lizability of the sperm.

Binding of hedamycin to deoxyribonucleic acid and chromatin of testis and liver.

The binding of the antibiotic hedamycin to DNA was evaluated by density gradient centrifugation in CsCl to determine the type I binding, which is essentially irreversible at high and low ionic strength. Exhaustive dialysis at low ionic strength was used to determine the sum of type I and type II binding (irreversible at low ionic strength but reversible at high ionic strength). The maximum ratio of hedamycin to DNA nucleotides (rf) is 0.1 for type I and 0.1 for type II binding to free DNA, but these ratios decrease to 0.07--0.08 in chromatin of rat liver and a testis fraction (spermatogonia plus primary spermatocytes). The rf for type I binding of hedamycin to monomeric nucleosomes of this testis fraction is considerably less than maximum binding to polymeric nucleosomes or chromatin, suggesting that hedamycin binds more effectively to "linker" DNA than to DNA attached to the core of the nucleosomes. Hedamycin binding to the chromatin of the spermatid fraction of testis is greatly decreased in comparison with chromatin of early stages; this correlates with the change from nucleohistones to nucleoprotamines at the middle to late spermatid stages of spermiogenesis.

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.

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 X 1 F1 was greatest in spermatid nuclei. On the other hand, the ratio X 3 F2b 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.

The ultrastructure of nuclear differentiation during spermiogenesis in the salmon

Progressive condensation of nuclear material occurs during spermiogenesis in the salmon. Nuclei of early spermatid stages contain randomly distributed, contorted fibers, similar in appearance, distribution, and mean diameter (175 A) to chromatin fibers of salmon somatic cell and spermatocyte nuclei. These fibers give way in nuclei of later spermatid stages to oriented, unusually thick fibers (with mean diameter 340 A), and to highly unusual, irregular, ribbonlike elements. In late spermatid stages nuclear fibers are no longer clearly visible; only short lengths of fibers and ribbonlike elements remain. Mature sperm nuclei are extremely electron-dense, showing little substructure. The unusually thick fibers and irregular ribbons which form during spermiogenesis in the salmon are clearly different from the typical fibers found in somatic cell nuclei and chromosomes and in the interphaselike nuclei of early spermatids. At least some of the striking structural changes which occur during nuclear condensation in salmon spermiogenesis probably are related to replacement of histones by protamine.

Zur frage einer kombinierten wirkung eines chemischen mutagens und strahlensterilisierter Nahrung im mutagenitäts- und reproduktionstest bei der Maus

The possible intensification of the mutagenic effect of cyclophosphamide (Endoxan) by the feeding of a radiation-sterilized diet (dose, 4·5 Mrad) was studied in 2000 NMRI/Han mice. In a dominant lethal test, males were pretreated with 100 mg Endoxan/kg body weight. The greatest sensitivity towards Endoxan was observed during the late-spermatid stage. No significant differences were detected between the control group (Endoxan plus non-irradiated diet) and the experimental group (Endoxan plus radiation-sterilized diet). In this test, radiation-sterilized feed showed no co-mutagenic effect when combined with Endoxan treatment. In a reproduction study of 7 months duration (continuous mating without lactation periods), the females were treated every 2 wk with 20 mg Endoxan/kg body weight. The decline in litter size with increasing number of litters (i.e. with advancing age of the females) was more pronounced after treatment with the chemical mutagen than in the untreated group. Increases in the frequency of abortions and in premature sterility resulted from Endoxan treatment. During the entire observation period, no effects from the intake of radiation-sterilized food were detected.

Methylation in vivo of guanine in the nucleic acids of rat testes by methyl methane sulphonate.

TREATMENT of male rats with a single intraperitoneal dose of 50 or 100 mg/kg body weight of methyl methane sulphonate results in infertility during the second and third week after the injection1. This infertility is caused not by aspermia but by a dominant lethal mutation in the sperm which are selectively affected at the late spermatid or early spermatozoa stage. The mutation does not interfere with the fertilization of the ova but the resulting embryos die in utero2. It has been assumed that this biological action of methyl methane sulphonate is connected with its properties as an alkylating agent. When a bacteriophage is inactivated by treatment with methyl methane sulphonate, guanine and adenine of the bacteriophage DNA are methylated to 7-methyl guanine, 3-methyl adenine, and 1-methyl adenine in proportion 78 : 20 : 2 (ref. 3). We have now found that an intravenous injection of methyl methane sulphonate methylates the 7 position of the guanine of DNA and RNA in rat testes.

X-ray induced mutability in male germ cells of Drosophila melanogaster

From the time the Drosophila melanogaster male is less than an hour old, sperm are continuously maturing, becoming motile, and entering the seminal vesicle, regardless of sexual activity. When a male, at any stage in its life history, is exposed to X-rays, all existing germ-cell stages are simultaneously irradiated. After irradiation, sperm will be sequentially produced by the male more or less in order of their stage of maturation at the time of irradiation, the first sperm samples being comprised of germ cells that were most mature at the time of irradiation. As the time interval between irradiation and the initial mating increases, the sperm samples consist of ever less homogeneous mixtures of cells that were at varying stages of maturity at the time of irradiation. If the male mates at a regulated pace after irradiation, sperm samples can be obtained that enable one to measure separately, and with reasonable accuracy, the mutability of mature sperm, intermediate and late spermatid stages, and early spermatids. Even so, unless each male in an experiment mates the same number of times each day, sampling of the various stages will become more and more asynchronous, as some males will produce sperm derived from stages quite different from those sampled at the same time from other males in the same experimental group. Then, if mutation frequencies are collected in terms of days after irradiation, they will not accurately reflect the mutability of any particular germ cell stage. When appropriate experimental procedures are followed, the mutability of mature, motile sperm is found to be as high, or higher, in male irradiations as in irradiations of inseminated females. The sex-linked recessive lethal mutation frequency characteristic of mature, motile sperm approaches 4% per 1000 R. Immotile sperm stages (intermediate and late spermatids) have a much lower mutability, less than 2% per 1000 R. Early spermatids again have a high mutability, probably exceeding that of motile sperm. The actual mutation frequency encountered at any time following irradiation reflects the proportion of the various kinds of irradiated germ cells in the sperm sample assayed. The precise proportion in any given sperm sample will be related to the age of the male at the time of irradiation, his previous mating history, the length of time that has elapsed between the irradiation and mating, and the number of inseminations that have ensued since the irradiation. The inherent mutability of male germ cells can be correlated with their physiological state at the time of irradiation. Thus, rapidly differentiating early spermatids are highly mutable and radiosensitive. Later, after the major structural modifications of the cell have been accomplished, the immotile intermediate and late spermatids exhibit a low mutability. Finally, in the terminal step in their differentiation the spermatids acquire motility, thereby becoming transformed into functional spermatozoa whose mutability is simultaneously reset to a high level.

The Effect of Temperature on the Enzyme Activities During Insect Spermatogenesis

SUMMARY1. The effects of cold and warm temperatures have been described. The optimum temperature at which the spermatogenesis takes place in the laboratory conditions is 35° C.2. The specimens at cold temperature show the spermatogonia and the spermatocytes indicating that the spermatogenesis has been initiated but is checked due to the inhibition of the enzyme activities. This phenomenon can again be revived if the specimens are transferred to the optimum temperature.3. In the high temperature conditions, the spermatogenesis continues till the late spermatid stage, but the latter does not transform into the final sperm due to the destruction of the enzymes.

The response of the immature testis of Drosophila to the mutagenic action of x-rays.

Drosophila melanogaster males were irradiated at various stages of pre-imaginal development, and mutation rates were compared between first broods of different age groups, and between successive broods in the same age group. The findings were correlated with observations on germ cell development in the larval and pupal testis. Sensitivity to the mutagenic effect of X-rays is low in all premeiotic stages. It increases suddenly and dramatically with the onset of meiosis, remains stationary through meiosis, and subsequently increases again up to a peak in the late spermatid stage when transformation into morphologically mature spermatozoa is taking place. This stage provides sperm for the first brood from males treated at about the middle of the pupal period, and mutation rate for a given dose of X-rays is here about 4 times as high as in mature sperm and about 1 1/2 times as high as during meiosis. Subsequently, mutation rate drops again gradually to the level characteristic for mature sperm. Germinal selection cannot account for any of these changes; but it may be the main cause for the slight increase in response which occurs during spermatogonial development.