Tests for urethane induction of germ-cell mutations and germ-cell killing in the mouse.

Urethane, a chemical that has given varied results in mutagenesis assays, was tested in the mouse specific-locus test, and its effect on germ-cell survival was explored. Altogether 32,828 offspring were observed from successive weekly matings of males exposed to the maximum tolerated i.p. dose of 1750 mg urethane/kg. The combined data rule out (at the 5% significance level) an induced mutation rate greater than 1.7 times the historical control rate. For spermatogonial stem cells alone, the multiple ruled out is 3.2, and for poststem-cell stages, 3.5. Litter sizes from successive conceptions made in any of the first 7 weeks give no indication of induced dominant lethality, confirming results of past dominant-lethal assays. That urethane (or an active metabolite) reaches germ cells is indicated by SCE induction in spermatogonia demonstrated by other investigators. Cytotoxic effects in spermatogonia are suggested by our finding of a slight reduction in numbers of certain types of spermatogonia in seminiferous tubule cross-sections and of a borderline decrease in the number of litters conceived during the 8th and 9th posttreatment weeks. The negative results for induction of gene mutations as well as clastogenic damage are at variance with Nomura's reports of dominant effects (F1 cancers and malformations) produced by urethane.

Do spermatogonial stem cells have a circadian rhythm?

Mitotic index was determined in whole mounts of segments of seminiferous tubules of (101 X C3Hf)F1 male mice at 3 hr intervals from 18.00 to 06.00 hours, and at hourly intervals from 08.00 to 16.00 hours. The highest frequency of metaphase-anaphase figures occurred at 10.00 and 11.00 hours, but was not significantly higher than for other times. Injection of 25 mu Ci 3H-TdR per mouse, followed 24 hr later by exposure to 300 rad X-rays and killing 207 hr after labelling was used to test for circadian rhythm in DNA synthetic activity of the long-cycling As spermatogonia. No significant effect of time of day was observed. Likewise, the number of undifferentiated spermatogonia scored 183 hr after 300 rad showed no effect of time of day. The testis therefore appears to have no circadian rhythm in mitotic activity. Stage of the cycle of the seminiferous epithelium, however, showed a significant effect on mitotic index of As spermatogonia and on DNA synthetic activity of undifferentiated spermatogonia. These data are compared with those for other organisms and tissues in respect to which properties of stem cells are general for all organisms and tissues and which are specific for spermatogonia.

Lack of effect of dibromochloropropane on the mouse testis.

A single intraperitoneal injection of (101 X C3Hf)F male mice with 110 mg/kg of 1,2-dibromo-3-chloropropane (DBCP) showed no effect on spermatogonial stem cells or on differentiating spermatogonia as measured by cell counts made 3, 5, and 8 days after injection.

The effect of ethyl-, methyl- and hydroxyethyl-nitrosourea on the mouse testis.

Hybrid male 101 X C3HF1 mice were given intraperitoneal injections of methyl-, ethyl- and hydroxyethyl-nitrosourea and killed 3-16 days later. All compounds were similar in that all differentiating spermatogonia from type A1 to early type B were killed by 50 mg/kg and higher doses of ENU and by 75 mg/kg MNU. Cells exposed in leptotene to 100 and 250 mg/kg ENU and 455 mg/kg HENU showed a delayed response with degeneration in pachytene 5 days later. Labeling prior to exposure to ENU indicated that the effect of stage of the mitotic cycle on sensitivity to cell killing is less marked than for radiation. This may be the explanation for the s-shaped mutation induction curve obtained with ENU in contrast to the humped dose-response curve observed for radiation.

Spermatogenic stage sensitivity to 6-mercaptopurine in the mouse.

Hybrid (101 X C3H)F1 male mice were given [3H]thymidine intraperitoneally, and 1 h later 150 mg/kg 6-mercaptopurine in 0.03 N NaOH. Autoradiography of testis sections showed that the rate of spermatogenesis was not altered, and the time of appearance of labeled spermatozoa in the ejaculate indicated that 6-mercaptopurine also had no effect on minimum sperm transport time. Labeled spermatozoa persisted in the ejaculate for a longer interval in 6-mercaptopurine-treated males than in controls, most likely as a result of oligospermia. Combined treatment with 150 R X-rays and 150 mg/kg 6-mercaptopurine gave an additive effect and demonstrated conclusively that the peak incidence of dead implants observed at 30.5 - 35.5 days can be attributed to cells treated as preleptotene spermatocytes and must result from genetic damage that is not cytologically detectable; previous work has shown that chromatic and isochromatid breaks at diakinesis-metaphase I occurred only on days 14 and 15 after 150 mg/kg 6-mercaptopurine. From the present experiments it is clear that these aberrations are not related to the dominant lethality at 30.5 - 35.5 days.

Synaptonemal complexes at premeiotic interphase in the mouse spermatocyte.

Male mice were injected intraperitoneally with 125 microCi (1 Ci = 3.7 X 10(10) becquerels) of [3H]thymidine at 1-hr intervals and killed 1 hr after the second injection. Testes were prepared for bright-field and electron microscopic autoradiography. Primary spermatocytes, identified by light microscopy to be at the premeiotic interphase stage, were found to be heavily labeled. Electron microscopic examination disclosed the coincidental occurrence of synaptonemal complexes and label within the nuclei of premeiotic interphase spermatocytes, indicating synapses of homologues had begun during the S phase. The significance of this finding for the traditional view of meiosis is discussed.

Follicular growth and atresia in the mouse.

Follicles were classified on the basis of the number of layers of follicle cells, the presence and degree of development of the zone pellucide, and the presence of an antrum. Formation of an antrum in follicles with less than 7 to 8 cell layers and/or presence of necrotic cells was considered indicative of degeneration. When classified in this manner, the data suggest that follicles and their contained oocytes are committed to either normal development or atresia by the time a third layer of granulosa cells is formed.

Timing of oocyte maturation in the mouse and its relevance to radiation-induced cell killing and mutational sensitivity.

Timing oocyte development by labeling the zona pellucida indicates that it takes 6 weeks (possibly a few days more) for a stage 3b oocyte to reach ovulation. Thus the shift in mutation frequency with time after irradiation occurs in an oocyte stage that is comparable in all mammals so far investigated, and in the mouse low-mutational sensitivity is not restricted to the arrested dictyate oocyte stage. Some oocytes with nuclear morphology similar to the arrested human oocytes give low mutation rates. Degree of chromosome condensation in early oocytes does not appear to be a reliable criterion of oocyte sensitivity to either cell killing or mutation induction, and genetic data on mouse oocytes may be more generally applicable than commonly thought.

Morphological and quantitative analysis of spermatogonia in mouse testes using whole mounted seminiferous tubules, I. The normal testes.

The spermatogonial populations in ten normal adult mice were analyzed using whole mounted seminiferous tubules. The undifferentiated A spermatogonia as well as the six generations of differentiating spermatogonia were clearly identifiable on whole mounts. Description plus quantitation of these cell types revealed that they behaved in essentially the same manner as their counterparts in the rat. Single undifferentiated A cells were classified as type As stem cell spermatogonia. They were distributed throughout the seminiferous epithelium, and by periodic mitoses, maintained their stock and furnished cells which would eventually differentiate. Although initially resembling the As spermatogonia, the progeny which were destined to differentiate were classified as type Aal spermatogonia because they were linked by cytoplasmic bridges, and because they usually underwent one or more synchronous mitotic divisions to form short chains of aligned cells. Ultimately, division of Aal cells were no longer seen, and the cells appeared to gradually acquire the typical morphological characteristics of A1 spermatogonia; these continued to differentiate according to the well-established pattern. It was concluded that the cyclic production of cohorts of A1 cells in this manner would ensure a continual supply of spermatogonia for differentiation.

Morphological and quantitative analysis of spermatogonia in mouse testes using whole mounted seminiferous tubules. II. The irradiated testes.

In adult male mice exposed to 300 R X-irradiation, the spermatogonial population was selectively killed except for the radioresistant type As stem cells. Type A spermatogonia were minimal two days after irradiation, when only 20% of the control population was present in stage 5-6; these were predominately single and paired undifferentiated cells. When multiple injections of 3HTdR were given between 2 and 3.5 days post-irradiation, 90-95% of these survivors in stages 4-6 became labeled. Enhanced proliferation of these stem cells, and at times when they were normally quiescent, led to restoration of all classes of spermatogonia by 11 days after irradiation. Several autoradiographic studies were undertaken to better characterize the radioresistant cells. In mice given single or multiple injections of 3HTdR prior to irradiation, there was appreciable retention of label by those type As spermatogonia that had originally incorporated 3HTdR in stages 2-4. This labeling pattern was identical to that of the long-cycling As stem cells in nonirradiated testes. Since the long-cycling As stem cells are thought to be characterized by a prolonged G1 or "A-phase" which is known to be a highly radioresistant portion of the cell cycle, it was clear why these cells could preferentially survive irradiation doses that killed other spermatogonial types. It was proposed that following germ cell depletion, as after irradiation injury, the long-cycling As survivors could be prematurely triggered from A-phase into DNA synthesis, thereby, initiating restoration of the germ cell population.