Role of cell cycle control in radiosensitization of mouse spermatogonial stem cells.

PURPOSE: To investigate the possible role of cell cycle arrest in the radiosensitization of mouse spermatogonial stem cells due to small conditioning X-ray exposures. MATERIALS AND METHODS: A 24 h fractionation interval between conditioning (1 Gy) and challenging (8 or 9 Gy) exposures was used. Two approaches were followed: the first in the Swiss random-bred wild-type mouse of the radiation-induced cell cycle arrest-evading agents 3-aminobenzamide (3-AB) and caffeine; and, second, using the C57BL/6 mouse of different p53 status. As biological parameter stem cell survival was analysed by the repopulation index (RI) method and chromosomal translocations were recorded using spermatocyte analysis at appropriate posttreatment periods. RESULTS: In the Swiss wild-type mouse, the application of 3-AB or caffeine significantly suppressed the sensitization of stem cells towards killing or translocation induction. In the C57BL/6 mouse, somewhat more variability in response was observed but no significant differences in sensitization between the p53 +/+, +/- or -/- mouse were recorded, suggesting no involvement of p53 in this process. CONCLUSIONS: The results indicate that p53-independent cell cycle regulation plays an important role in the radiosensitization of mouse spermatogonial stem cells.

Effect of hypothyroidism on satellite cells and postnatal fiber development in the soleus muscle of rat.

The effect of hypothyroidism, induced by 4-propyl-2-thiouracil, on muscle satellite cells in vivo and in vitro, and on postnatal muscle fiber development in the soleus muscle of rats during the first 40 days of postnatal life was analyzed. The proliferative activity of satellite cells was determined by means of bromodeoxyuridine incorporation. Creatine kinase activity was used as a marker for differentiation. In vivo, hypothyroidism resulted in smaller fibers in which the amount of sarcoplasm remained in balance with the number of myonuclei. The in vivo labeling data of satellite cells did not indicate a decreased proliferative activity, but the in vitro experiments showed that the hypothyroid rat muscles contained fewer satellite cells that were less active in proliferation and differentiation at the start of culture. Despite this, the bromodeoxyuridine signal increased in time at a similar rate as that in control cultures. From this and because the cells resembled control cells in their response to bFGF, we conclude that hypothyroid satellite cells remain responsive to proliferation stimuli. However, in hypothyroid cultures, the activity of creatine kinase is lower, even at longer culture times. We therefore conclude that hypothyroid status affects muscle precursor cells mainly by depressing their ability to differentiate and fuse with existing myofibers.

Prednisone can protect against exercise-induced muscle damage.

In an experimental animal exercise model we tested whether daily administration of prednisone prevents the development of mechanically induced muscle fibre damage. Six-week-old rats were treated with different doses of prednisone ranging from 1 to 50 mg/kg body weight per day or with placebo, for 8 days. On day 6 of treatment the rats were forced to run for 2 h on a level treadmill. Two days after exercise morphological damage in the soleus muscles was quantified using light microscopy and a semi-automatic image analysis system. Creatine kinase (CK) activity was measured before exercise (day 5) and directly after exercise (day 6). The expression of dystrophin in a placebo group and in a group that received 5 mg prednisone/kg body weight per day with and without performing exercise was studied with Western blotting. The effect of prednisone on fibre type distribution was determined with an antibody against fast myosin and the effect of prednisone on the proliferative activity of muscle satellite cells was studied using bromodeoxyuridine (BrdU) immunohistochemistry. Exercise-induced muscle fibre damage varied in a dose-dependent way. In the placebo group the mean (SEM) damaged muscle fibre area was 4% (1%). The groups that received low doses of prednisone, 1 or 2.5 mg/kg per day, showed a similar level of muscle damage. However, with 5 mg prednisone/kg per day the amount of muscle fibre damage [mean (SEM)] was significantly reduced to 1.4% (0.5%) (P

Satellite cell activation after muscle damage in young and adult rats.

BACKGROUND: Level exercise leads to focal structural damage in muscle fibers and to an increase of creatine kinase in the blood. We questioned whether it also induces activation of young and adult muscle satellite cells toward proliferation. METHODS: Rats of two different ages, 6 and 16 weeks, were forced to run on a level treadmill and killed at different time intervals. The temporal profile, up to 3 weeks, of muscle damage was investigated by quantification of the focally disturbed fiber area in longitudinal sections of the m. soleus. Bromodeoxyuridine (BrdU) was injected before death to determine the labeling index of satellite cells. Labeled and unlabeled satellite cells, myonuclei, and fibers were counted in cross sections of the belly part of the muscles. RESULTS: The muscle fiber damage differed in both amount and temporal profile between young and older animals. Damage was already visible immediately after running. However, whereas in the younger animal the amount of damage increased gradually in time until 8% at 48 hours and disappeared to almost control levels at 1 week after running, in the older animals the amount of damage was lower but remained present for at least 2 weeks. The cell kinetic data on both groups showed a proliferation response of satellite cells throughout the muscle. The effects were most pronounced in the older rats. In these rats a large increase of the labeling index was found between 24 hours and 1 week, whereas the total number of satellite cells was consistently higher from 2 days on until 2 weeks after running. In the younger animals roughly the same time pattern was observed. CONCLUSION: Since the damage differed in amount and time between the two age groups, we conclude that the quick and huge proliferation response is due to leakage of mitogenic factors through small membrane disruptions that are generated during the exercise itself.

The induction of chromosomal damage and cell killing in mouse spermatogonial stem cells following combined treatments with hydroxyurea, 3-aminobenzamide and X-rays.

To analyze in more detail the relation between the sensitivity of spermatogonial stem cells to killing and the induction of genetic damage, mature male mice received combined treatments with hydroxyurea (HU), 3-amino-benzamide (3-AB) and X-rays. Stem cell killing was determined using the repopulation index method and translocations were studied via spermatocyte analysis. HU was administered at 16 or at 48 h before further treatment in order to create stem cell populations with different sensitivities in which the translocation induction and stem cell killing could be studied and compared. The sensitivities for cell death and genetic damage appeared to be strongly correlated: at 16 h after HU significantly higher values were found than at 48 h or in controls without HU pretreatment. By using 3-AB in the treatment schedules we were able to investigate whether the sensitization of stem cells towards cell death and genetic damage is the outcome of a radiation- or drug-induced G1 delay. The effect of 3-AB was most pronounced at 16 h after HU. This confirms that at this interval a large fraction of stem cells is in G1. Our data therefore indicate that all treatments that induce an enrichment of G1 cells also result in a sensitization of stem cells to cell killing or the induction of mutagenic damage.

Repeated interruptions of the testicular blood flow do not have long-term effects on spermatogenesis in the ram.

The effect of repeated interruptions of the testicular blood flow on spermatogenesis was studied in mature Texel rams. Reversible interruption of the blood flow was achieved by an inflatable occluder, placed around the testicular artery at the level of the spermatic cord. In eight testes the blood flow was successfully interrupted six times for 1 h within 3 weeks and in 14 testes nine times for 1 h within 3 weeks. Nine weeks after the last blood flow interruption spermatogenesis was evaluated in histological sections of the testes. Both after six and nine blood flow interruptions a qualitatively complete epithelium was found in at least 90% of the seminiferous tubules. Cell counts in stages VII and VIII of the spermatogenic cycle revealed a slight decrease of spermatocytes and spermatids in the tubules with a complete epithelium after nine occlusions, which was only statistically significant for Preleptotene Spermatocytes. After six occlusions the numbers of all cell types were at or even slightly above control levels. These results show that repeated periods of ischaemia for 1 h do not result in conspicuous long-term damage to spermatogenesis.

Protection of spermatogenesis against cytotoxic effects of two chemotherapeutic drugs by temporary testicular blood flow interruption in the ram.

Temporary interruption of the testicular blood flow for 1 h after injection of cytostatic drugs has a protective effect on spermatogenesis. This was shown in experiments in which spermatogenesis was evaluated at four weeks after a single intravenous injection of Adriblastina (ADR; doxorubicin hydrochloride) or Mitomycin-C-kyowa (MIT). Interruption of the blood flow was performed by inflation of an occluder implanted around the testicular artery. The animals were killed and histological sections prepared 4 weeks after treatment. In all drug-treated animals spermatids were near absence and spermatocytes were decreased in number. Therefore, even after occlusion of the blood flow, the drug doses were high enough to kill not only large numbers of differentiating spermatogonia but also stem cells. The response of the stem cells to the treatments was evaluated by counting the numbers of A spermatogonia per 100 Sertoli cells in the different groups. Normal numbers of these cells were found after both MIT and ADR, indicating that the stem cell population had responded to the initial cell loss by extra proliferation. However, significantly higher numbers of A spermatogonia were found in the drug-treated animals in which the testicular blood flow was interrupted for 1 h. This indicates that occlusion of the blood flow to the testis for 1 h results in a faster recovery of spermatogenesis than after drug treatment alone.

Effects of X-irradiation and adriamycin on quiescent and proliferating cells of the seminal vesicle in the castrated mouse.

The sensitivity of resting and proliferating cells of the seminal vesicle to X-irradiation and adriamycin has been investigated. Stimulation with testosterone propionate (250 micrograms/day) was started 11 days after castration in BALB/c mice. X-rays (2.5-7.5 Gy total body irradiation) and intraperitoneal injections of adriamycin (4-16 mg/kg body weight) were administered at various times before or after induction of proliferation by testosterone injection. The DNA contents and the weights of the seminal vesicles were determined at 4 days after the start of stimulation. A Do for X-rays of about 10 Gy was found for the seminal vesicle epithelium. For both X-irradiation and adriamycin no significant differences in sensitivity were observed between quiescent (Go) and proliferating (G1; S) seminal vesicle cells.

Dose-response studies on the spermatogonial stem cells of the rhesus monkey (Macaca mulatta) after X irradiation.

Studies of the dose response of the spermatogonial stem cells in the rhesus monkey were performed at intervals of 130 and 160 days after graded doses of X irradiation. The D0 of the spermatogonial stem cells was established using the total numbers of the type A spermatogonia that were present at 130 and 160 days after irradiation and was found to be 1.07 Gy; the 95% confidence interval was 0.90-1.34 Gy.

Protective effect of hypoxia in the ram testis during single and split-dose X-irradiation.

Spermatogonial stem-cell survival in the ram was studied after single (6 Gy) and split-dose (2 x 3 Gy, interval 21-24 h) X-irradiation both under normal and hypoxic conditions. Hypoxia was induced by inflation of an occluder implanted around the testicular artery. The occluders were inflated about 10 min before irradiation and deflated immediately after. Stem-cell survival was measured at 5 or 7 weeks after irradiation by determination of the Repopulation Index (RI) in histological testis sections. The RI-values after fractionated irradiation were only half those after single dose irradiation. Hypoxia had a protective effect on the stem-cell survival. After split-dose irradiation under hypoxic conditions two times more stem cells survived than under normal oxic conditions; the RI-values increased from 34% (oxic) to 68% (hypoxic). This effect of hypoxia was also found after single dose irradiation where the RI-values increased from 68% (oxic) to 84% (hypoxic). The development of the epithelium in repopulated tubules was also studied. Under hypoxia, a significantly higher fraction of tubules with complete epithelium was found after single (38 vs. 4%) as well as after split-dose irradiation (12 vs. 0%).

The cell cycle of spermatogonial colony forming stem cells in the CBA mouse after neutron irradiation.

In the CBA mouse testis about 10% of the stem cell population is highly resistant to neutron irradiation (D0, 0.75 Gy). Following a dose of 1.50 Gy these cells rapidly increase their sensitivity towards a second neutron dose and progress fairly synchronously through their first post-irradiation cell cycle. From experiments in which neutron irradiation was combined with hydroxyurea it appeared that in this cycle the S-phase is less radiosensitive (D0, 0.43 Gy) than the other phases of the cell cycle (D0, 0.25 Gy). From experiments in which hydroxyurea was injected twice after irradiation the speed of inflow of cells in S and the duration of S and the cell cycle could be calculated. Between 32 and 36 hr after irradiation cells start to enter the S-phase at a speed of 30% of the population every 12 hr. At 60 hr 50% of the population has already passed the S-phase while 30% is still in S. The data point to a cell cycle time of about 36 hr, while the S-phase lasts 12 hr at the most.

Reversible harmless interruption of testicular blood supply in the ram.

An effective method of interrupting testicular blood flow temporarily and repeatedly in the ram has been developed. Blockade of flow has been achieved mechanically by an inflatable occluder placed around the testicular artery at the level of the spermatic cord. The effect of the blockade on total testicular blood supply was investigated using Doppler flowmetry and a percutaneous Xenon-133 injection method. With both approaches, the blood flow changes after inflation or deflation of the occluders could be estimated satisfactorily. A substantial decrease of testicular blood flow was achieved in eight of the 10 testes with inflated occluders. However, there were indications that in the remaining two testes blockade of the arterial flow was not complete. After deflation of the occluders, blood flow was restored rapidly and completely in all testes. Macro- and microscopic examinations revealed no long-term damage to the testis after blood flow interruptions lasting 30 or 60 minutes.

Cell-cell interaction between rat Sertoli cells and mouse germ cells in vitro.

An antiserum against rat germ cell membranes was prepared, and after absorption with protein extracts of rat liver and kidney and mouse testis, this antiserum reacted only with rat germ cell membranes and juxtanuclear vesicles in rat Sertoli cells. Germ cell-free rat Sertoli cell monolayers were cultured in vitro. Freshly isolated mouse germ cells adhered to these monolayers within 1 h. After a minimum of 3 days of such a co-culture, immunofluorescence and immunoblotting revealed that the mouse germ cells had obtained rat-antigenic determinants in their membranes. Our results indicate that this appearance of rat-specific antigens on mouse germ cells is specific and inducible.

Strain differences in the response of mouse testicular stem cells to fractionated radiation.

The survival of spermatogonial stem cells in CBA and C3H mice after single and split-dose (24-hr interval) irradiation with fission neutrons and gamma rays was compared. The first doses of the fractionated regimes were either 150 rad (neutrons) or 600 rad (gamma). For both strains the neutron survival curves were exponential. The D0 value of stem cells in CBA decreased from 83 to 25 rad upon fractionation; that of C3H stem cells decreased only from 54 to 36 rad. The survival curves for gamma irradiation, which all showed shoulders, indicated that C3H stem cells had larger repair capacities than CBA stem cells. However, the most striking difference between the two strains in response to gamma radiation was in the slopes of the second-dose curves. Whereas C3H stem cells showed a small increase of the D0 upon fractionation (from 196 to 218 rad), CBA stem cells showed a marked decrease (from 243 to 148 rad). The decreases in D0 upon fractionation, observed in both strains with neutron irradiation and also with gamma irradiation in CBA, are most likely the result of recruitment or progression of radioresistant survivors to a more sensitive state of proliferation or cell cycle phase. It may be that the surviving stem cells in C3H mice are recruited less rapidly and synchronously into active cycle than in CBA mice. Thus, it appears that the strain differences may be quantitative, rather than qualitative.

Growth and differentiation of spermatogenetic colonies in the mouse testis after irradiation with fission neutrons.

The longitudinal outgrowth of spermatogenetic colonies arising from stem cells that survived neutron doses of 150, 300, and 350 rad was studied up to 30 weeks in histological sections of CBA mouse testes. Two methods were used: (1) the repopulation index (RI) as a measure of the length of total colonies per testis and (2) measurement of the individual length of all colonies in serially sectioned testes 4 and 15 weeks after 300 rad and 15 weeks after 350 rad. The mean initial growth of the colonies is linear up to 8, 15, and 20 weeks after 150, 300, and 350 rad, respectively. Although after these doses the mean initial colony growth rate did not differ significantly (about 27 microns/day), both methods showed that the colonies grow about 20% slower after 350 rad. Screening of individual colonies revealed a great variation in colony length per testis and a higher frequency of short colonies with higher neutron doses. Counting of colonies after 300 rad showed that all surviving stem cells had started to form a colony within 4 weeks after irradiation. The development of spermatogenetic cells to mature spermatozoa was studied after 100, 150, 300, and 350 rad in sections of repopulating tubules used for RI determination as well as in serial sections of individual colonies. Although after 300 and 350 rad spermatogenetic cell types beyond the stage of young spermatocytes reappeared 1 week late, we found no great disturbances in the regular reappearance of the successive spermatogenetic cell types after irradiation. However, from the study of individual colonies it appeared that colonies differ widely in their development even within one testis. Moreover, the frequency of less developed colonies was higher after 350 rad than after 300 rad. Our data suggest that this retardation in the reappearance of further developed cells is caused by a delay in the production of developed cells in spermatogonia in an increasing fraction of the colonies after higher neutron doses. Even in fully developed colonies the production of differentiating spermatogenetic cell types was subnormal after 300 and 350 rad. This was caused by an extensive cell degeneration in the colonies as well as by a tendency of the undifferentiated and/or A1-spermatogonial population to increase its own number at the cost of the production of further developed cells.