Syntheses of nucleic acids during spermatogenesis in Nereis diversicolor (annelida polychaeta): a quantitative autoradiographic study

The development of DNA and RNA synthesis in the germ cell population was studied after a 3H-thymidine or 3H-uridine pulse at each stage of spermatogenesis. The autoradiographic results show that the first sign (after 3 days in vitro) of cellular changes is an increase in RNA synthesis which reaches a maximum at day 5. DNA replication (premeiotic S phase) occurred at day 7, then cells entered meiotic prophase (day 9). Meiotic divisions and spermiogenesis occurred after 11 days. Silver grain counts permit the conclusion that RNA synthesis is clearly higher during premeiotic interphase (days 3-7) than during spermatogonial proliferation (day 0). It appears therefore that male meiotic differentiation in Nereidae is accompanied by increased RNA synthesis.     

Premeiotic germ cell defect in seminiferous tubules of Atm-null testis

Lifelong spermatogenesis is maintained by coordinated sequential processes including self-renewal of stem cells, proliferation of spermatogonial cells, meiotic division, and spermiogenesis. It has been shown that ataxia telangiectasia-mutated (ATM) is required for meiotic division of the seminiferous tubules. Here, we show that, in addition to its role in meiosis, ATM has a pivotal role in premeiotic germ cell maintenance. ATM is activated in premeiotic spermatogonial cells and the Atm-null testis shows progressive degeneration. In Atm-null testicular cells, differing from bone marrow cells of Atm-null mice, reactive oxygen species-mediated p16(Ink4a) activation does not occur in Atm-null premeiotic germ cells, which suggests the involvement of different signaling pathways from bone marrow defects. Although Atm-null bone marrow undergoes p16(Ink4a)-mediated cellular senescence program, Atm-null premeiotic germ cells exhibited cell cycle arrest and apoptotic elimination of premeiotic germ cells, which is different from p16(Ink4a)-mediated senescence.     

Culture of intact Sertoli/germ cell units and isolated Sertoli cells from Squalus testis: I. Evidence of stage-related functions in vitro

As part of an ongoing program of research using the testis of the dogfish shark (Squalus acanthias) to characterize morphologic and functional changes during spermatogenesis, we have developed procedures for culturing intact spermatocysts (germ cell/Sertoli cell clones) and isolated Sertoli cells from premeiotic, meiotic, and postmeiotic stages of development. Phase contrast and light microscopy confirmed the stage and cellular composition of spermatocysts and showed that they retained their closed, spherical configuration for at least 15 d in culture. Stage-related variations in [3H]thymidine incorporation (premeiotic much greater than meiotic = postmeiotic) were observed, a pattern that was the same quantitatively and qualitatively after one or seven days of culture. [3H]Leucine-labeled protein synthesis was twofold greater in cultures with premeiotic spermatocysts than in cultures with more mature stages, whether medium or cysts were analyzed. Sertoli cells isolated from spermatocysts of different stages differed in size, shape, cytological appearance, ability to form flattened monolayers, and rate of DNA synthesis. One day after seeding, [3H]thymidine labeling of Sertoli cells corresponded to the pattern obtained with intact spermatocysts (premeiotic much greater than meiotic = postmeiotic); however, 7 days in culture effected a 40- to 200-fold increase in this parameter and altered the stage-dependent pattern (premeiotic = meiotic greater than postmeiotic). Also, when [3H]leucine-labeled macromolecules secreted by Sertoli cells from premeiotic versus meiotic stages were analyzed by polyacrylamide gel electrophoresis (PAGE), banding patterns differed. Initial results demonstrate the feasibility and potential of this in vitro system for studying qualitative and quantitative changes during spermatogenesis.     

Initiation and kinetic profiles of spermatogenesis in the frog, Runa esculenta (Amphibia)

Spermatogenesis in Rana esculenta is initiated during metamorphic climax and mature spermatozoa are present in froglets 45 days old. Cytological analysis of cell populations shows that some of the primary spermatogonia may lie dormant for brief intervals of time. Timing analysis of the process of spermatogenesis, in adults and in developing Frog larvae maintained at approximately 18°C, was investigated by different methods. The results are remarkably similar. Although perfect synchrony of the developing cells within a single germinal cyst is not the rule, a uniform rate of advancement of germinal cysts of the same stage of development, in most of the seminiferous tubules of a testis is evident. The duration of the secondary spermatogonial divisions is five to six days, involving five or six mitotic cycles, each cycle lasting approximately 24 h. The premeiotic S-phase, and phases of leptotene, zygotene, pachytene, diplotene+secondary spermatocytes, and spermiogenesis each have a duration, respectively, of six, two, six, twelve, two and seven days. The duration of spermatogenesis, from a "committed" primary spermatogonium to the formation of spermatozoa is 41 days.     

Cyclic AMP and genetic sterility of a male restricted (H re /+) mutant of Rattus

Restricted (H ^re /+) male rats marked by a coat color pattern have normal testes at birth. By 9 days postpartum, testes of the mutant animals are smaller than normal and by approximately 90 days of age the animals are sterile. The genetically sterile testes are totally devoid of spermatogonial cells, spermatocytes, spermatids, and spermatozoa, with only Sertoli cells remaining in the seminiferous tubules. Cyclic AMP concentrations in the whole testes (and the seminiferous tubules) of the mutant males are approximately 10–35% greater than in testes of control males when tested at intervals from 5 to 120 days of age. The possible role of excess cyclic AMP in reducing the rate of mitotic division of spermatogonial cells while enhancing differentiation of spermatogonial cells into spermatozoa is discussed. Such a change in the respective rates of mitotic and meiotic divisions would ultimately deplete the mutant testes of all spermatogonial cells.     

Cell proliferation and differentiation kinetics during spermatogenesis inHydra carnea

Summary Spermatogenesis inHydra carnea was investigated. The cell proliferation and differentiation kinetics of intermediates in the spermatogenesis pathway were determined, using quantitative determinations of cell abundance, pulse and continuous labelling with³H-thymidine and nuclear DNA measurements. Testes develop in the ectoderm of male hydra as a result of interstitial cell proliferation. Gonial stem cells and proliferating spermatogonia have cell cycles of 28 h and 22 h, respectively. Stem cells undergo four, five or six cell divisions prior to meiosis which includes a premeiotic S+G2 phase of 20 h followed by a long meiotic prophase (22 h).Spermatid differentiation requires 12–29 h. When they first appear, testes contain only proliferating spermatogonia; meiotic and postmeiotic cells appear after 2 and 3 days, respectively and release of mature sperm begins after 4 days. Mature testes produce about 27,000 sperm per day over a period of 4–6 days: about 220 gonial stem cells per testis are required to support this level of sperm differentiation. Further results indicate that somatic (e.g. nematocyte) differentiation does not occur in testes although it continues normally in ectodermal tissue outside testes. Our results support the hypothesis that spermatogenesis is controlled locally in regions of the ectoderm where testes develop.     

Cycle of the seminiferous epithelium and its duration in the zebu, Bos indicus

The cycle of the seminiferous epithelium was studied in Nelore zebu bulls 4–6 years old. The stages of the cycle were determined according to the shape and position of spermatid nuclei and the presence of meiotic divisions in cross-sections of seminiferous tubules. The relative frequencies of stages 1 to 8 were, respectively: 31.3 ± 0.5, 12.2 ± 0.7, 21.2 ± 0.5, 8.8 ± 0.6, 4.2 ± 0.4, 5.7 ± 0.6, 6.3 ± 0.5 and 10.3 ± 0.5. The duration of the cycle was estimated by autoradiography using tritiated thymidine injected directly into the testes. The mean duration of one cycle was estimated to be 14.0 ± 0.4 days.     

KINETICS OF CELL RENEWAL, CELL MIGRATION AND CELL LOSS IN THE HAIRLESS MOUSE DORSAL EPIDERMIS

Forty hairless mice were given injections of tritiated thymidine every 4th hour during 10 days. At 24 hr intervals groups of four mice were killed. The numbers of labelled basal and differentiating cells were determined by autoradiography with a stripping film technique. To determine the background activity skin sections from uninjected control mice were subjected to the same stripping film procedure. Another group of hairless mice was given one single pulse labelling with tritiated thymidine. The number of labelled mitoses was scored for 12 hr after the injection. At 10, 12 and 15 hr after the injection, the numbers of labelled basal and differentiating cells were also determined. A mathematical model of cell population kinetics in the epidermis has been suggested. The results of different simulations on this model were compared with the observed results. The curve of mean grain counts under continuous labelling increased from day to day with two well-defined plateaux. The percentage of all labelled cells increased rapidly up to the 3rd day, and thereafter the curves gradually flattened off. When basal cells and differentiated cells were considered separately the labelling index of the basal cells increased rapidly for the first 3 days and then flattened off at the 100% level on the 5th day. The labelling index of the differentiating cells was low during the first 3–4 days. Then a steep increase in the percentage of labelled differentiating cells was seen, but the curve flattened off again close to the 100 % level after the 7th day. The labelled mitosis curve had its maximum 5 hr after the thymidine injection. The curve fell again to almost zero at 12 hr. Ten, 12 and 15 hr after the injection, 6, 7 and 7% respectively of the labelled cells were found in the spinous layer. It was concluded that three grains over each nucleus could be used as lower limit for considering a cell as labelled. On this basis, tritiated thymidine injections every 4th hour can be considered as continuous labelling.     

Seasonal patterns of ornithine decarboxylase activity and levels of polyamines in relation to the cytology of germinal cells during spermatogenesis in the sea star, Asterias vulgaris

The activity of ornithine decarboxylase (ODC) and levels of polyamines were measured in the testes of Asterias vulgaris collected throughout an annual spermatogenic cycle. Samples of the testes were prepared for light and electron microscopy to observe the associated changes in the cytology of germinal cells. The specific activity of ODC increased at the onset of testicular growth, decreased during the coldest period of the winter when testicular growth was minimal, and increased again early in the spring when testes grew maximally. Increased activity of ODC resulted in increased levels of polyamines and was correlated with either mitotic or meiotic germinal cell divisions, or both. Spermine levels were always greater than putrescine, followed by spermidine. Highest levels of polyamine synthesis coincided with the onset of spermatogonial proliferation during the fall and with the period of meiotic differentiation and spermiogenesis in the spring. Mid-summer (July) testes were small (0.3-0.5 gonad index (GI)) and contained amitotic spermatogonia arrested in G(1) of the cell cycle. Mitotic and pre-meiotic testes (October/November) increased slightly in size (0.3-1.4 GI) and contained actively dividing spermatogonia, most of which differentiated into primary spermatocytes. Testes from February and March were large (1-6.75 GI), but the proliferative status of their spermatogonia and primary spermatocytes varied. Spermatogonia and primary spermatocytes from early February testes were not dividing. In testes obtained in March, both spermatogonial mitosis and meiosis of spermatocytes resumed, coincident with increased field water temperatures.     

Rate of loss of tritiated thymidine label in basal cells in mouse epithelial tissues

A subpopulation of epithelial cells which retains a tritiated thymidine label (termed label-retaining cells, LRCs) has been previously demonstrated in skin and oral mucosae of mice and hamsters. To examine the rate of decrease in the number of LRCs and the changes in degree of labelling, young mice were labelled with tritiated thymidine and the rate at which label was diluted from basal keratinocytes assessed for up to 90 days. The number of LRCs in each tissue examined decreased from 15 to 90 days after labelling with the epidermal tissues maintaining a higher percentage of LRCs than the oral mucosae. Grain counts for LRCs in each tissue at each time period indicated that the number of silver grains overlying LRCs also decreased with time. The observed decrease in numbers of LRCs and the change in their degree of labelling with time suggest that such cells divide slowly, a property associated with stem cells.     

Rate of Loss of Tritiated Thymidine Label In Basal Cells In Mouse epithelial tissues

A subpopulation of epithelial cells which retains a tritiated thymidine label (termed label-retaining cells, LRCs) has been previously demonstrated in skin and oral mucosae of mice and hamsters. to examine the rate of decrease in the number of LRCs and the changes in degree of labelling, young mice were labelled with tritiated thymidine and the rate at which label was diluted from basal keratinocytes assessed for up to 90 days. the number of LRCs in each tissue examined decreased from 15 to 90 days after labelling with the epidermal tissues maintaining a higher percentage of LRCs than the oral mucosae. Grain counts for LRCs in each tissue at each time period indicated that the number of silver grains overlying LRCs also decreased with time. the observed decrease in numbers of LRCs and the change in their degree of labelling with time suggest that such cells divide slowly, a property associated with stem cells.     

Analysis of cell types identifiable in air-dried cell preparations of human testis

The mixed cell population of the testicular epithelium has been studied in air-dried cell preparations obtained from a testicular biopsy. Observed cell types are defined, quantified and assigned to cell stages of the spermatogenic cycle. Studies with tritiated thymidine helped to categorize the spermatogonial cell types. Variation in cell size within cell categories, variation in frequency of cells in different categories within individuals, and variation in frequency of cells within categories between individuals were subjected to quantitative analysis.     

Cycle and duration of the seminiferous epithelium in puma (Puma concolor)

Puma or sussuarana (Puma concolor) is the second largest feline in the American continent and has an ample latitudinal distribution in very diverse habitats. In relation to its conservation status, the puma is considered an extinction-threatened species. The study of the testis morphology and the spermatogenic process in a species is fundamental for establishing the physiologic patterns that will make possible the selection of the protocols for assisted reproduction. A number of peculiarities associated with the reproductive biology of specific species such as the duration of spermatogenic process can be used to determine the frequency of sperm collection. Nine adult male pumas maintained in captivity were used to determine the relative frequency of stages in the seminiferous epithelium cycle. Three of them received intra-testicular injections of 0.1ml tritiated thymidine to determine the duration of the seminiferous epithelium cycle, and were subjected to biopsy 7 days later. The cycle of the seminiferous epithelium in puma was didactically described into eight stages by the tubular morphology method. The total duration of one seminiferous epithelium cycle in puma was calculated to be 9.89 days, and approximately 44.5 days are required for development of spermatozoon from spermatogonia. The duration of spermiogenesis, prophase and other events of meiosis were 14.08, 15.20 and 1.79 days, respectively. The relative frequency of the pre-meiotic, meiotic and post-meiotic phases were 3.98, 1.79 and 4.12 days, respectively.     

Characterization and inducibility of hsp 70 proteins in the male mouse germ line

The properties and inducibility of the heat shock protein 70 (hsp 70) gene products were examined during differentiation of mouse testicular cells by one and two-dimensional gel electrophoresis and immunoblotting. Low levels of the 72- and 73-kD heat shock proteins normally found in mouse cell lines were detected in the mouse testis. A novel isoform with a relative molecular mass of 73 kD (called 73T) was also observed, in the presence or absence of heat shock. 73T was shown to be produced by germ cells since it was not detected in testes from mutant mice devoid of germ cells. Furthermore, 73T was found only in adult mouse testicular cells, not in testes from animals that lack meiotic germ cells. 73T was synthesized in enriched cell populations of both meiotic prophase and postmeiotic cells, but was not inducible by in vitro heat shock. In the adult testis, low levels of the bona fide 72-kD heat-inducible (hsp72) were induced in response to elevated temperatures. In contrast, in testes from animals in which only somatic cells and premeiotic germ cells were present, there was a substantial induction of hsp 72. It is suggested that hsp 72 is inducible in the somatic compartment and possibly in the premeiotic germ cells, but not in germ cells which have entered meiosis and which are expressing members of the hsp 70 gene family in a developmentally regulated fashion.     

Analysis of Cell Types Identifiable In Air-Dried Cell Preparations of Human Testis

Abstract. The mixed cell population of the testicular epithelium has been studied in air-dried cell preparations obtained from a testicular biopsy. Observed cell types are defined, quantified and assigned to cell stages of the spermatogenic cycle. Studies with tritiated thymidine helped to categorize the spermatogonial cell types. Variation in cell size within cell categories, variation in frequency of cells in different categories within individuals, and variation in frequency of cells within categories between individuals were subjected to quantitative analysis.     

Stage-specific damage to synaptonemal complexes and metaphase chromosomes induced by X rays in male mouse germ cells

Synaptonemal complexes reveal mutagen-induced effects in germ cell meiotic chromosomes. This study was aimed at characterizing relationships between damage to synaptonemal complexes and metaphase I chromosomes following radiation exposure at various stages of spermatogenesis. Male mice were irradiated with doses of 0, 2, or 4 Gy, and spermatocytes were harvested at times consistent with earlier exposures as spermatogonial stem cells, preleptotene cells (premeiotic DNA synthesis), or meiotic prophase cells. After stem-cell exposure, twice as many rearrangements were observed in synaptonemal complexes as in metaphase I chromosomes. Irradiation during premeiotic DNA synthesis resulted in dose-related increases in synaptonemal complex breakage and rearrangements (including novel forms) and in metaphase chromosomal aberrations. Following prophase exposure, various types and levels of damage to synaptonemal complexes and metaphase chromosomes were observed. Irradiation of zygotene cells led to high frequencies of chromosome multivalents in metaphase I without a correspondingly high level of damage in preceding prophase synaptonemal complexes. Thus irradiation of premeiotic and meiotic cells results in variable relationships between damage to synaptonemal complexes and metaphase chromosomes. Interpretations of these relationships are based upon what is known about both radiation clastogenesis and the structural/temporal relationships between synaptonemal complexes at prophase and chromosomes at metaphase I of meiosis.     

SOME MORPHOLOGIC AND KINETIC STUDIES OF THE DEVELOPING ERYTHROID CELLS OF THE COMMON GOLDFISH, CARASSIUS AURATUS

Developing erythroid cells of the goldfish Carassius auratus were obtained from kidney prints and from smears of the peripheral blood. All preparations were stained with the May-Grunwald Giemsa technique. Developing cells were divided into six different stages. The criteria used to stage the cells were degree of chromatin condensation, degree of basophilia, nuclear:cytoplasmic ratio, and cell shape. The morphology of the maturation sequence for erythroid cells in this organism was similar to that found by other workers in other non-mammalian vertebrates. Fish received intraperitoneal injections of tritiated thymidine, tritiated uridine or tritiated leucine so that the stages involved in DNA synthesis, RNA synthesis and protein synthesis could be determined by means of autoradiography. For the tritiated thymidine studies the per cent labeled cells per stage from four different series receiving 0.5, 1.0, 3.0 or 6.0 μCi/g body weight were pooled, since subjecting the average per cent labeled cells per stage at the lowest and at the highest dosages to Student's t-test showed no significant differences. In all four series the fish were killed 2 hr, 12 hr and daily, 1–8 days post-injection. The ³H-TdR studies showed that stages I-IV were engaged in DNA synthesis; they also showed that about 5 days were required for the stage V cell to become a mature erythrocyte (stage VI cell). Tritiated uridine was injected at a dosage of 5.0 μCi/g body weight and animals were killed 1/2, 1, 3 and 6 hr post-injection. Grain counts showed that stages I-IV are engaged in RNA synthesis and that the rate of this synthesis decreased as maturation proceeded. Tritiated leucine was administered at a dosage of 5.0 μCi/g body weight, and fish were killed 45 min and 3 hr post-injection. Grain counts indicate that stages I-V are engaged in the synthesis of protein (assumed to be globin). The fact that DNA and RNA synthesis ceased with stage IV cells while protein synthesis continued into stage V cells indicated that the mechanism responsible for protein synthesis in stage V cells was produced at an earlier stage and was self-sustained for about 5 days.     

Intracellular antigenic changes associated with meiosis in the orthopteran Stauroderus scalaris.

Monoclonal antibodies have been prepared against purified pachytene cells from grasshopper testes. Immunoblotting and immunofluorescence analyses identified those monoclonal antibodies which showed specificity for antigens in pachytene cells. Several antigenic changes were found to be associated with meiotic cells. Five monoclonal antibodies detected antigens which were located in the cytoplasm of premeiotic cells but were nuclear during meiosis. One monoclonal antibody showed a discrete cytoplasmic fluorescent pattern in meiotic, but not in premeiotic, cells. Another bound specifically to the nuclei of some epithelial cells at the base of follicles in mature testes.     

Seminiferous epithelium cycle and its duration in capybaras (Hydrochoerus hydrochaeris).

Although capybara is the largest rodent in the world and largely distributed in Central and South America, there is no report in the literature concerning the cycle of seminiferous epithelium in this species. In the present study, the length of spermatogenic cycle was estimated using intratesticular injections of tritiated thymidine. Animals were sacrificed at 1 h, 8 days, and 17 days after injections. The duration of one spermatogenic cycle in capybaras is 11.9 +/- 0.1 days (SEM). Spermatogenesis was estimated to last 53.6 days, when considering that the total duration of spermatogenesis takes about 4.5 cycles of seminiferous epithelium. The approximate life span of primary spermatocytes is 19.1 days, while spermiogenesis lasts 16.7 days. Staging in capybaras was based on the spermatid nuclei shape and location of spermatids, named tubular morphology method, which consists of 8 stages in all species. The relative stage frequencies in capybaras, based on the analysis of approximately 200 cross sections of seminiferous tubule for each of the ten animals were as follow: stage 1: 14.0 +/- 1.5%; stage 2: 15.1 +/- 1.0%; stage 3: 15.7 +/- 1.1%; stage 4: 14.6 +/- 1.1%; stage 5: 8.7 +/- 0.7%; stage 6: 7.0 +/- 0.7%; stage 7: 9.4 +/- 0.9%; stage 8: 15.5 +/- 1.0%. The pre-meiotic, meiotic and post-meiotic phases relative frequencies were 44.8%, 14.6% and 40.6%, respectively. Compared to most rodents investigated so far, the duration of spermatogenesis in capybaras is relatively long.     

FAILURE TO IDENTIFY A SPERMATOGONIAL CHALONE IN ADULT IRRADIATED TESTES

The adult irradiated rat testis was used as a model system to confirm the existence of a spermatogonial chalone. Rats were given 330 rad whole body ⁶⁰Co irradiation, a dose which selectively destroys most of the spermatogonial population except for the radioresistant A_s stem cells. 11 days after irradiation, when spermatogonial numbers were minimal, the rats were injected with a testicular or liver extract prepared from normal adult rats, or with saline. Each group received a total of four injections given at 4 hr intervals. 2 hr before death, the animals were injected with [³H]TdR. Testicular DNA was isolated and the incorporation of [³H]TdR was determined. The mean ± s.e. ct/min per μg DNA in rats given testicular extract (9·38 ± 0·94) was no different than in those receiving liver extract (10·43 ± 2·01) or saline (7·23 ± 0·69). Autoradiographic studies indicated that variability in counts within or between groups could be attributed to variations in the number of pre-leptotene spermatocytes which incorporated [³H]TdR for the meiotic divisions. Quantitatively, there were no differences between groups in terms of the numbers of A spermatogonia, their labelling indices, or mitotic activity. Therefore, the presence of a spermatogonial chalone could not be demonstrated using crude extracts from normal testes in this irradiated model.