Effect of age on the sensitivity of mouse spermatogonia to mutagenic action]

Male BALB/c mice were whole body exposed to 0, 100, 200 or 300 R of X-rays or were given an i.p. injection of 5 mg/kg of thio-TEPA at 3 months or at 12 months of age. One hundred days after treatment the testes were examined for the presence of reciprocal translocations induced in spermatogonia and detectable in the dividing spermatocytes. Our results show that ages does not influence the yield of scorable chromosome rearrangements.     

Cytogenetic effects of cyclophosphamide on mouse spermatogonia

Summary NMRI mice were treated with single doses of cyclophosphamide (Cytoxan) and spermatogonia were analysed for chromosome aberrations at various time intervals after treatment.The maxima of aberrations were found 24 hrs p.i. Chromatid type aberrations were observed exclusively. About half of the aberrations consisted of chromatid interchanges, 92% of which exchanging short arm fragments close to the centromeric region.The lack of meiotic multivalents in diakinesis-metaphase I after treatment of spermatogonia stem cells with cyclophosphamide in the study of leonard and Linden (1972) is discussed.

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Zusammentassung NMRI-Mäuse erhielten einmalig eine Applikation von Endoxan in verschieden hoher Dosierung. Zu verschiedenen Zeiten nach der Behandlung wurden Tiere getötet und die Spermatogonien analysiert.Das Maximum der induzierten Effekte lag bei 24 Std p.i. Es gelangten ausschließlich Chromatidentypaberrationen zur Beobachtung, die annähernd zur Hälfte aus Chromatidenaustauschen bestanden. Dabei handelte es sich überwiegend (92%) um Segmentaustausche im zentromernahen Bereich.Der fehlende Nachweis von Multivalenten in der Diakinese-Metaphase I nach Behandlung spermatogonialer Stammzellen mit Cyclophosphamid (Leonard u. Linden, 1972) wird diskutiert.

     

Action of mitomycin C on mouse spermatogonia

The induction of chromosome aberrations in mouse spermatogonia and bone marrow cells by treatment with Mitomycin (MC) was tested. The following dosages were used: 3.5; 1.75; 0.35; and 0.035 mg/kg body weight. Chromatid interchanges and terminal deletions were induced in both tissues. Regarding the chromosome damage, spermatogonia seemed to be more sensitive to the test substance than bone marrow cells.The aberrations observed were considered to represent the cause of dominant lethals induced in spermatocytes after treatment with MC by others. The squash technique adapted for examination of mitoses of mouse spermatogonia proved to be a useful tool in testing potential chemical mutagens.     

Strain differences in the radiosensitivity of mouse spermatogonia

The radiosensitivity of spermatogonia was found to be greater by up to a factor of 2 in C3H mice than in B6D2F1 mice, whether assessed for the highly sensitive spermatogonia (types A2 to In) or the much more resistant clonogenic spermatogonia which repopulate tubules. The latter were similarly resistant in the B6D2F1 hybrid and in the DBA2 parent, but were much more sensitive in the C57BL parent strain. A difference in sensitivity by up to a factor of 2 results in a variation by a factor of 10 or more in the level of survival of clonogenic cells after high doses. This variation is also observed when comparing data in the literature from different authors using various strains of mice. Using the radiosensitizer misonidazole, it was shown that hypoxia did not play a major role in the lesser sensitivity demonstrated in B6D2F1 mice. The variation in sensitivity is similar to the range reported in the literature for reciprocal translocations.     

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.     

Cytogenetic effects of cyclophosphamide on mouse spermatogonia.

NMRI mice were treated with single doses of cyclophosphamide (Cytoxan) and spermatogonia were analysed for chromosome aberrations at various time intervals after treatment. The maxima of aberrations were found 24 hrs p.i. Chromatid type aberrations were observed exclusively. About half of the aberrations consisted of chromatid interchanges, 92% of which exchanging short arm fragments close to the centromeric region. The lack of meiotic multivalents in diakinesis-metaphase I after treatment of spermatogonia stem cells with cyclophosphamide in the study of Leonard and Linden (1972) is discussed.     

Nonhomologous end joining of complementary and noncomplementary DNA termini in mouse testicular extracts

Mammalian somatic cells are known to repair DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ) and homologous recombination (HR); however, how male germ cells repair DSBs is not yet characterized. We have previously reported the highly efficient and mostly precise DSB joining ability of mouse testicular germ cell extracts for cohesive and blunt ends, with only a minor fraction undergoing terminal deletion [Mutat. Res. 433 (1999) 1]; however, the precise mechanism of joining was not established. In the present study, we therefore tested the ability of testicular extracts to join noncomplementary ends; we have also sequenced the junctions of both complementary and noncomplementary termini and established the joining mechanisms. While a major proportion of complementary and blunt ends were joined by simple ligation, the small fraction having noncleavable junctions predominantly utilized short stretches of direct repeat homology with limited end processing. For noncomplementary ends, the major mechanism was "blunt-end ligation" subsequent to "fill-in" or "blunting", with no insertions or large deletions; the microhomology-dependent joining with end deletion was less frequent. This is the first functional study of the NHEJ mechanism in mammalian male germ cell extracts. Our results demonstrate that testicular germ cell extracts promote predominantly accurate NHEJ for cohesive ends and very efficient blunt-end ligation, perhaps to preserve the genomic sequence with minimum possible alteration. Further, we demonstrate the ability of the extracts to catalyze in vitro plasmid homologous recombination, which suggests the existence of both NHEJ and HR pathways in germ cells.     

Effect of temperature and the role of testicular descent on post-natal testicular androgen production in the mouse.

Testes from mice aged 3, 15, 25, 30 or 60 days were incubated under basal conditions or in the presence of hCG. One testis from each animal was incubated at 37 degrees C while the contralateral testis was incubated at 32 or 34 degrees C. During development total androgen production in response to hCG (at 32 degrees C) showed a marked increase between 15 and 30 days. The major androgens secreted at this time were testosterone and 5 alpha-androstane-3 alpha,17 beta-diol. There was little change in total androgen production between 30 and 60 days but by 60 days testosterone was the dominant androgen. Both basal and hCG-stimulated androgen production were temperature sensitive. These effects were most pronounced at 30 and 60 days with androgen production significantly inhibited at 37 degrees C. To examine the role of testicular descent in regulating steroidogenesis animals were rendered unilaterally cryptorchid at 19 days of age. At 25 days, when descent is normally completed in the mouse, there was no significant difference in steroidogenesis between scrotal and abdominal testes. By 30 days, however, the steroidogenic potential of the abdominal testis was significantly lower than that of the scrotal testis. These results show that testicular steroidogenesis is sensitive to temperature changes around the time of testicular descent, although descent itself is not required to achieve an adult level of steroidogenesis. The results also show, however, that testicular descent is required to maintain the adult level of steroidogenesis.     

Effects of FGF9 on embryonic Sertoli cell proliferation and testicular cord formation in the mouse

Proliferation and cord formation by embryonic Sertoli cells are pivotal events involved in testis morphogenesis. A number of growth factors have been implicated in mediating these events. However, the exact level of involvement and importance of each as yet remains elusive. We have adopted an in vitro approach to assess developing mouse Sertoli cells, whereby they are cultured in the presence or absence of fibroblast growth factor (FGF9) and/or extracellular matrix (ECM) gel, since previous studies have shown that ECM gel aids Sertoli cell differentiation. The present findings corroborate this effect, but in addition demonstrate that in the presence of FGF9 (10 ng/ml), cells undergo greater proliferation than those cultured on gel alone. They also display a differentiated epithelial phenotype, with appositional contact of cell membranes in cord-like aggregations. In addition we have shown that cultured Sertoli cells generally express a smaller truncated, nuclear form of the FGFr3, although in the presence of FGF9 and absence of gel, the larger, cytoplasmic form of the receptor is also expressed. Immunolocalisation of FGFr3 in Sertoli cells of whole testes revealed a temporal expression pattern profile, with high levels being abundant in the embryonic testicular cords and at puberty, but an absence in adult Sertoli cells. Our findings suggest that FGF9 plays an important role in proliferation and organisation of embryonic Sertoli cells during testis morphogenesis.     

Effect of testosterone on testicular steroidogenesis in the hypogonadal (hpg) mouse

Previous studies have shown that androgens have direct inhibitory effects on steroidogenesis in active Leydig cells. It is not clear what effect androgens have on inactive Leydig cell either through direct action on the cell itself or indirectly through stimulation of Sertoli cell activity. The hpg mouse has undetectable levels of circulating gonadotrophins and the gonads fail to develop post-natally. The effect of androgen treatment on testicular steroidogenesis and morphology was examined in these animals. Treatment with testosterone propionate for two weeks significantly increased testicular and seminal vesicle weight. Seminiferous tubules showed marked development in androgen-treated animals, indicating increased Sertoli cell activity, but the abnormal Leydig cell morphology of the hpg testis was unchanged. Androgen production per testis in vitro was low in control hpg animals and remained unaffected by treatment with androgen. Similarly, the pattern of [3H]pregnenolone metabolism was not significantly affected by androgen treatment. The androgen content of the testis was higher in androgen-treated animals but this could be accounted for by uptake of administered steroid from the circulation. It is concluded that androgens have no direct trophic effect on Leydig cells and that stimulation of Sertoli cell activity is not, in itself, sufficient to affect Leydig cell function.     

High-resolution light microscopic characterization of mouse spermatogonia

Characteristics of spermatogonia were determined in the C57BL/6J strain mouse using high-resolution light microscopy of plastic-embedded tissues and identifying cells during stages of the spermatogenic cycle. The frequency of expecting each spermatogonial cell type was a major factor in identifying and categorizing various cell types. Although numerous characteristics were described, several major differences were noted in spermatogonial cell types. The group comprising A(s), A(pr), and A(al) spermatogonia could be differentiated based primarily on mottling of heterochromatin throughout the nucleus in the absence of heterochromatin lining the nuclear envelope. The A(1) cells displayed finely granular chromatin throughout the nucleus and virtually no flakes of heterochromatin along the nuclear membrane. The A(2) through A(4) spermatogonia contained progressively more heterochromatin rimming the nucleus. Intermediate-type spermatogonia displayed flaky or shallow heterochromatin that completely rimmed the nucleus. Type B spermatogonia showed rounded heterochromatin periodically along the nuclear envelope. Use of gray-scale histograms allowed objective quantification of nuclear characteristics and showed a logical shift in the gray scale to a narrower and darker profile, from four cell types leading to A(1) cells. The ability to differentiate spermatogonial types is a prerequisite to studying the behavior and kinetics of the earliest of the germ cell types in both normal and abnormal spermatogenesis.