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The mitochondrial proteins involved in spermatogenic cells apoptosis in zebrafish after carbon ion radiation (CIR) were screened. The relative biological effectiveness (RBE) of CIR in zebrafish testes was investigated. Apoptosis of testicular cells was measured within 24 hr following 1 and 4 Gy CIR. Immunoblotting was used to assess the levels of mitochondrial apoptotic proteins in testes, and proliferative and apoptotic spermatogenic cells were detected by immunofluorescence after CIR. Label-free quantitative (LFQ) and parallel reaction monitoring-based target proteomics (PRM) were combined to screen and validate differential mitochondrial proteins in testes between 4 Gy and control groups at 24 hr after CIR. The RBE of CIR in zebrafish testes was 1.48 ± 0.04, and induction of apoptosis by CIR was higher than that of X-rays in testicular cells. Mitochondrial apoptotic pathways play a crucial role in spermatogenic cells apoptosis after CIR, with 60 differential mitochondrial proteins identified. Among 20 target proteins, 12 were significantly upregulated, 2 were significantly downregulated in the 4 Gy CIR group. The results of PRM were consistent with label-free analysis. This is the first study to screen the differential mitochondrial proteins and provide useful information to understand the underlying mechanisms of spermatogenic cell apoptosis in zebrafish following CIR., (© 2019 Wiley Periodicals, Inc.)

Kisspeptin (Kp) expression in testis has caused most of the recent research surveying its functional role in this organ. This peptide influences spermatogenesis and sperm capacitation, so it is considered as a regulator of reproduction. Kp roles exert through hypothalamic/pituitary/gonadal axis. We aimed to evaluate direct roles for Kp on proliferation and differentiation of spermatogonial cells (SCs) when the cells are cocultured with somatic cells. Somatic cells and SCs were isolated from adult azoospermic and newborn mice and then enriched using a differential attachment technique. After the evaluation of identity and colonization for SCs, the cells were cocultured with somatic cells, and three doses of Kp (10 -8 -10 -6  M) was assessed on proliferation (through evaluation of MVH and ID4 markers) and differentiation (via evaluation of c-Kit and SCP 3 , TP 1, TP 2 , and, Prm 1 markers) of the coculture system. Investigations were continued for four succeeding weeks. At the end of each level of testosterone in the culture media was also evaluated. We found positive influence from Kp on proliferative and differentiative markers in SCs cocultured with somatic cells. These effects were dose-dependent. There was no effect for Kp on testosterone level. From our findings, we simply conclude that Kp as a neuropeptide for influencing central part of reproductive axis could also positively affect peripheral processes related to spermatogenesis without having an effect on steroidogenesis., (© 2018 Wiley Periodicals, Inc.)

Macrophage migration inhibitory factor (MIF) is a multifunctional molecule. MIF was originally identified as a T-cell-derived factor responsible for the inhibition of macrophage migration. In testicular tissue of adult rats, MIF is constitutively expressed by Leydig cells under physiological conditions. The aim of this study was to examine MIF levels in testicular homogenates from different aged mice, and the capacity of Sertoli cells to produce it. We also examined MIF involvement in spermatogonial cell migration. Similar levels of MIF protein were detected in testicular homogenates of mice of different ages (1-8-week-old). However, the RNA expression levels of MIF were high in 1-week-old mice and significantly decreased with age compared to 1-week-old mice. MIF was stained in Sertoli, Leydig cells, and developed germ cells in the seminiferous tubules. Isolated Sertoli cells from 1-week-old mice stained to MIF. Cultures of Sertoli cells from 1-week-old mice produced and expressed high levels of MIF which significantly decreased with age. MIF was localized in the cytoplasm and nucleus of Sertoli cell cultures isolated from 1-week-old mice; however, it was localized only in the cytoplasm and branches of cultures isolated from 8-week-old mice. MIFR was detected in GFRα1 and Sertoli cells. MIF could induce migration of spermatogonial cells, and this effect was synergistic with glial cell-line neurotrophic factor. Our results show, for the first time, the capacity of Sertoli cells to produce MIF under normal conditions and that MIFR expressed in GFRα1 and Sertoli cells. We also showed that MIF induced spermatogonial cell migration. J. Cell. Physiol. 232: 2869-2877, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Micro RNAs play important roles during mammalian spermatogenesis, but the function(s) of specific miRNAs remain largely unknown. Here, we report that miR-100 is predominantly expressed in undifferentiated murine spermatogonia, including spermatogonial stem cells (SSCs). We utilized a miRNA mimic and inhibitor to knock down and overexpress miR-100 in cultured SSCs, respectively, finding that the miR-100 promotes the proliferation of SSCs in vitro. Furthermore, signals promoting SSC maintenance induced, whereas retinoic acid repressed, expression of miR-100. Stat3 expression was modulated by miR-100, with increased transcript and protein abundance in the presence of the miR-100 inhibitor versus reduced protein levels following miR-100 overexpression. Stat3 silencing also mimicked the reduced SSC proliferation phenotype associated with elevated miR-100 levels. Importantly, Stat3 silencing rescued the anti-proliferation capacity of miR-100 inhibitor on cultured SSCs. Given that the Stat3 3' untranslated region was not repressed by pre-miR-100 in a standard luciferase reporter assay, we suggest that miR-100 promotes SSC proliferation by indirect regulation of Stat3., (© 2017 Wiley Periodicals, Inc.)

D-aspartate (D-Asp) is an endogenous amino acid present in vertebrate tissues, with particularly high levels in the testis. In vivo studies indicate that D-Asp indirectly stimulates spermatogenesis through the hypothalamic-pituitary-gonadal axis. Moreover, in vitro studies have demonstrated that D-Asp up-regulates testosterone production in Leydig cells by enhancing expression of the steroidogenic acute regulatory protein. In this study, a cell line derived from immortalized type-B mouse spermatogonia retaining markers of mitotic germ cells (GC-1) was employed to explore more direct involvement of D-Asp in spermatogenesis. Activity and protein expression of markers of cell proliferation were determined at intervals during incubation in D-Asp-containing medium. D-Asp induced phosphorylation of ERK and Akt proteins, stimulated expression of PCNA and Aurora B, and enhanced mRNA synthesis and protein expression of P450 aromatase and protein expression of Estrogen Receptor β (ERβ). These results are the first demonstration of a direct effect of D-Asp on spermatogonial mitotic activity. Considering that spermatogonia express the NR1 subunit of the N-Methyl-D-Aspartic Acid receptor (NMDAR), we suggest that their response to D-Asp depends on NMDAR-mediated activation of the ERK and Akt pathways and is further enhanced by activation of the P450 aromatase/ERβ pathway., (© 2015 Wiley Periodicals, Inc.)

Concerns have been raised about the possible role of the phytoestrogen genistein and the xenoestrogen Bisphenol A (BPA) as endocrine disruptors. In the present study, we examined the effects of fetal exposure to genistein and BPA on the Mitogen-activated protein kinase (MAPK) pathway and on testicular cell populations in neonatal and adult rat testes. At postnatal day (PND) 3, genistein (0.1-10 mg/kg/day) and BPA (1-200 mg/kg/day) induced Raf1 and Erk1/2 mRNA and protein increases in testes, mainly in Sertoli cells. No changes were seen for Mek1. At PND60, Erk1/2 protein expression remained robust in Sertoli cells and in some spermatogonia. Raf1 was predominant in Leydig cells while Mek1 was expressed strongly in spermatogonia, and they were both expressed in pachytene spermatocytes. No consistent change was seen in these proteins at PND60. Transient effects were observed on germ cell populations, while the only remaining effect on adult testicular cells was an increase in Leydig cell number. Rats exposed in utero to the two compounds did not present significant changes in circulating testosterone levels, suggesting normally functioning adult Leydig cells. Furthermore, Sertoli cell numbers were not affected by exposure to genistein and BPA. Finally, around 10% of genistein and BPA exposed rats were sterile, whereas all control rats were fertile. These data suggest that fetal exposure to genistein and BPA exerts transient effects in rat testes and that the changes observed at PND3 did not correlate with relevant changes in germ cell populations, Leydig cell function, or fertility in the adult., ((c) 2009 Wiley-Liss, Inc.)

The fetal and neonatal development of male germ cells (gonocytes) is a poorly understood but crucial process for establishing fertility. In rodents, gonocytes go through two phases of proliferation accompanied by apoptosis and separated by a quiescent period during the end of fetal development. P63 is a member of the P53 gene family that yields six isoforms. We detected only the p63 protein and no p53 and p73 in the nucleus of the gonocytes of mouse testes. We report for the first time the ontogeny of each p63 mRNA isoform during testis development. We observed a strong expression of p63gamma mRNA and protein when gonocytes are in the quiescent period. In vitro treatment with retinoic acid prevented gonocytes from entering the quiescent period and was correlated with a reduced production of p63gamma isoform mRNA. We investigated the function of p63 by studying the testicular phenotype of P63-null mice. P63 invalidation slightly, but significantly increased the number of gonocytes counted during the quiescent period. As P63-null animals die at birth we used an original organ culture that mimicked neonatal in vivo development to study further the testicular development. P63 invalidation resulted in a sharply increased number of gonocytes during the culture period due to a decrease in spontaneous apoptosis with no change in proliferation. P63 invalidation also caused abnormal morphologies in the germ cells that were also found in P63(+/-) adult male mice. Thus, p63 appears as an important regulator of germ cell development.

Etoposide is a podophyllotoxin semiderivative that is used in a variety of chemotherapy treatments, including therapy for children tumors. This drug promotes the formation of a ternary DNA-topoisomerase II-etoposide complex that triggers apoptosis. The purpose of this work was to analyze the occurrence of apoptosis in the seminiferous epithelium of prepubertal, pubertal, and adult rats treated with 10, 20, and 40 mg/Kg of etoposide during the prepubertal phase, as well as the role of apoptosis in etoposide-induced testicular damage. The rat testes were fixed in Bouin's liquid, and the apoptotic cells were quantified by means of the hematoxylin and eosin (H&E) technique (all groups) and the terminal dUTP nick end labeling (TUNEL) method (prepubertal groups only). The results obtained from both the H&E and TUNEL methods showed an increased frequency of apoptosis in the seminiferous epithelium of treated animals, except for the subgroup that received the 10-mg/Kg dose and was sacrificed 12 hr after the treatment and for the etoposide-treated pubertal group, that did not show cells suggesting apoptosis during H&E analysis. The labeled cells were mainly primary spermatocytes and differentiated spermatogonia. The prepubertal rats showed an etoposide-dose-dependent diminution of differentiated spermatogonia. Etoposide treatment during the prepubertal phase increases the frequency of apoptosis in the seminiferous epithelium, and causes serious harm to male fertility. 2004., (Copyright 2004 Wiley-Liss, Inc.)

There are always more evidences indicating that 17beta-estradiol (E(2)) is necessary for normal male fertility. We have used a nonmammalian vertebrate model (the lizard Podarcis s. sicula) to investigate the regulation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) activity in the testis during the annual sexual cycle and to study whether E(2) exerts a role in the spermatogenesis through ERK1/2 activity. Immunocytochemistry analysis shows that ERK1/2 proteins are present in the nucleus of the spermatogonia (SPG), and in primary (I) spermatocytes (SPC). The annual E(2) profile shows a progressive increase during the active spermatogenesis (from April to June) and a peak in the month of August (spermatogonial mitosis). In parallel, ERK1/2 (molecular weight 44 and 42 kDa, respectively) are highly phosphorylated during the period of active spermatogenesis and in post-refractory period (August) compared with the winter stasis (from November to March). Present results demonstrate that E(2) treatment induces spermatogonial proliferation, possibly via the activation of ERK1/2, and this effect is counteracted by the antiestrogen ICI 182-780., (Copyright 2002 Wiley-Liss, Inc.)

The initial purpose of the study was to determine the potential of acetaldehyde (Ace) to increase the rate of sister-chromatid exchanges (SCEs) in mouse spermatogonia. We tested four doses of Ace (from 0.4 to 400.0 mg/kg), including a negative and a positive control group (distilled water and cyclophosphamide, respectively). The results showed that all tested doses were SCE inducers. The highest tested dose increased the control level more than three times. Also, the cumulative frequencies of SCEs per cell were higher in the Ace-treated animals than in the control cells. Ace is transformed into acetate through the enzyme aldehyde dehydrogenase, a process that may be blocked by disulfiram (Dis) generating the accumulation of Ace. The second purpose of the study was to determine if the administration of Dis (150 mg/kg) could increase the SCE rate produced by non-genotoxic doses of Ace. (0.004 and 0.04 mg/kg). The animals treated with the two doses of Ace alone showed no increase in the frequency of SCEs; also, Dis by itself was not an SCE inducer. However, the groups of animals previously treated with Dis showed an increase of 31 and 60% with respect to the values obtained with the two doses of Ace alone. Furthermore, the cumulative frequencies of SCEs per cell were higher in the animals administered with both compounds together than in those treated with them separately. These results suggest the need to extend this type of study to other models., (Copyright 2002 Wiley-Liss, Inc.)

Responses to genotoxic agents vary not only among organisms, test systems, and cellular stages, but also between sexes; little, however, is known about the mutagenic consequences of chemical exposures to female germ cells. In this study, the mutagenicity of N-ethyl-N-nitrosourea (ENU) was analyzed in female germ cells of Drosophila melanogaster using the recessive-lethal test and the vermilion system, which simultaneously generates information on induced mutation frequency and mutation spectrum. ENU was mutagenic in all stages of oogenesis, although there were differences among the stages. In mature and immature oocytes, ENU-induced mutations in the vermilion locus were 43.5% A:T-->G:C transitions, 39.1% A:T-->T:A transversions, 8.7% G:C-->A:T transitions, and 8.7% A:T-->C:G transversions, indicating that the most important premutagenic lesions induced by this chemical are O(4)-ethylthymine and O(2)-ethylthymine. The low frequency of mutation involving O(6)-ethylguanine (i.e., G:C-->A:T transitions) could be a consequence of the repair of these lesions by O(6)-methylguanine DNA methyltransferase. Comparison of these results with those previously obtained in male germ cells stresses the importance of the repair activity of the analyzed cells, because the mutation spectrum in female germ cells was similar to the spectrum obtained with repair-proficient spermatogonial cells and different from repair-deficient postmeiotic cells. The results also indicate that studies with female germ cells could be an alternative to the use of premeiotic male germ cells, especially when the analysis of these cells is difficult or almost impossible and when studies of in vivo DNA repair in premeiotic germ cells are performed., (Copyright 2002 Wiley-Liss, Inc.)

At the present time, in spite of recent advances, knowledge about the factors regulating germ cell proliferation in the teleost testis is limited. This study was designed to investigate, in vitro, the ability of various hormones, growth factors, and steroids to influence the proliferation of trout spermatogonia (Go) present in mixed cultures of somatic and germ cells prepared from testes, either prespermatogenetic or spermatogenetic. The tested molecules were usually present for the duration of culture (4.5 days) and 3H-thymidine (3H-Tdr) for the last day in culture. In our cell culture conditions, homologous gonadotropin I (tGTH-I) and growth hormone (tGH) moderately stimulated 3H-Tdr incorporation by Go, with ED50 equal to 5.5 +/- 3.0 and 1.8 +/- 0.4 ng/ml respectively. Insulin growth factor I (rhIGF-I) and fibroblast growth factor (rhFGF-2) stimulated 3H-Tdr incorporation by Go from spermatogenetic testes only, with ED50 equal to 16.2 +/- 9.3 and 2.4 +/- 0.3 ng/ml respectively. The effects of the most efficient concentrations of rhIGF-I combined with those of either tGTH-I or tGH were additive. Seventy to one hundred microM suramin stimulated 3H-Tdr incorporation by Go from testes at all maturation stages and this effect was additive with that of tGTH-I. We assume that this effect of suramin could result from the inhibition of an unidentified antimitogenic factor. No effect was observed with homologous prolactin, human epidermal growth factor, activin A and B, transforming growth factor-beta1, testosterone, 11-ketotestosterone, 17beta-estradiol, pregnenolone, 11beta-hydroxyprogesterone, and 22-hydroxycholesterol. In conclusion, our in vitro results suggest that GTH-I, GH, IGF-I, and FGF-2, are potent in situ modulators of the proliferative activity of trout Go at the time of induction, speeding up, then slowing down spermatogenesis, through direct or indirect additive and/or antagonistic influences., (Copyright 1999 Wiley-Liss, Inc.)

Unlike in higher vertebrates, in fish it is not known whether the nerve supply of the testis can influence testicular functions or not. In addition to neurotransmitters, nerve terminals may release ATP and adenosine in the extracellular medium. On the assumption that these molecules might be released by fibers innervating the teleost testis, it is possible that they participate in the control of testicular function and, maybe, in the control of spermatogonia (Go) proliferation. This study addresses this issue. We have investigated the ability for extracellular ATP and adenosine to influence the in vitro incorporation, either basal or GTH-, IGF-I- and suramin-stimulated, of 3H-thymidine (3H-Tdr) by trout Go. Mixed suspensions of somatic and germ cells prepared from testes, which were immature or spermatogenetic, were cultured usually for 4.5 days in the presence or not of the tested molecules; 3H-Tdr was added during the last day in culture. In our cell culture conditions, 25 to 250 microM adenosine, ATP, ADP, and AMP stimulated the 3H-Tdr incorporation by Go from prespermatogenetic testes and from testes starting spermatogenesis, in a dose-dependent way. The effect of these molecules decreased when the testes were more advanced in spermatogenesis and it became inhibiting when the testes were in mid-spermatogenesis. Five'-N-ethylcarboxamidoadenosine (NECA) was as potent as adenosine in stimulating or inhibiting 3H-Tdr incorporation, while R-N6-(2-phenylisopropyl)adenosine (R-PIA) always had a marked inhibiting effect. Adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS; 25-200 microM), a non-hydrolysable analogue of ATP, which had no effect on Go from prespermatogenetic testes (collected October-February) and from testes in advanced spermatogenesis, stimulated 3H-Tdr incorporation by Go from testes at the beginning of spermatogenesis very efficiently. The order of potency of the different ATP analogues was as follows: ATPgammaS > ATP congruent with alpha,beta-methylene-ATP > UTP > 2-methylthio-ATP. These data suggest that A2 adenosine receptors and P2 receptors would be present on unidentified testicular cells. The stimulating effect of adenosine/ATP was additive with that of either GTH-I or IGF-I or suramin when the cells were from testes at the beginning of spermatogenesis, but adenosine suppressed their effect when the cells were from testes in mid-spermatogenesis. In conclusion, our results suggest that in the trout extracellular adenosine and ATP are able to influence the in vitro proliferation of Go, and are potential candidates for mediating the possible influence of the nervous system on the induction, speeding up, then slowing down of spermatogenesis., (Copyright 1999 Wiley-Liss, Inc.)

In order to evaluate the pUR288-plasmid transgenic mouse model, utilizing the bacterial lacZ gene as the mutational target, radiation-induced mutagenesis was primarily analyzed in spermatogonial stem cells. A combined hydroxyurea (HU)-X-ray treatment protocol was used, known to sensitize dramatically the induction of mutations in endogenous genes. In the testes of untreated animals, a mutant frequency of 6.7 +/- 4.4 x 10(-5) was found. In animals treated with HU or X ray alone, moderate elevations were seen (factors of about 4 and 2 over untreated animal values). In testes of mice having received the HU + X-ray combination treatment, a mutant frequency of 63.0 +/- 36.1 x 10(-5) was found. The results obtained showed a good quantitative correlation between endogenous genes and the transgene, indicating the suitability of pUR288 transgenic mice for also efficiently recording radiation-induced genetic damage. Radiosensitization, seen in spermatogonial stem cells, was not observed in other studied organs such as spleen, brain, or lung., (Copyright 1999 Wiley-Liss, Inc.)

An autoradiographic procedure was used to measure unscheduled DNA synthesis (UDS, DNA repair synthesis) in spermatogonial and postspermatogonial cell stages of mice after treatment with two doses of N-ethyl-N-nitrosourea (ENU) and N-methyl-N-nitrosourea (MNU). Significant levels of UDS were measured in type A spermatogonia, meiotic spermatocytes, round spermatids, and early elongating spermatids but not in mature spermatids. The extent of UDS varied according to the germ cell stage and the dose. At equimolar concentrations, MNU was more efficient than ENU in eliciting a UDS response in all germ cells. After ENU treatment, type A spermatogonia showed the highest UDS response, while round and elongating spermatids showed the lowest. After MNU treatment, pachytene spermatocytes exhibited the highest UDS response while type A spermatogonia showed the lowest. The high UDS response of type A spermatogonia to ENU parallels the well-known high mutational sensitivity of spermatogonia to this chemical. Similarly, the high UDS response observed in meiotic spermatocytes and early spermatid stages after MNU treatment correlates with the high mutational sensitivity of postspermatogonial stages to MNU. Thus, the present results, like the specific locus mutation studies, indicate that ENU and MNU each has a unique effect on the spermatogenic cells. This effect is likely due to the different mechanism of action of ENU and MNU at the level of DNA and also to the physiological differences between different germ-cell stages. Teratogenesis Carcinog. Mutagen. 19:339-351, 1999. Published 1999 Wiley-Liss, Inc.

In order to explore the significance of DNA methylation in proliferation and differentiation of germ cells in testis, 5-aza,2'-deoxyCytidine (5-azaCdR), a hypomethylating agent, was administered in vivo to neonatal mice having only spermatogonial (premeiotic) cells. End-labeling of the MspI, HpaII, and HhaI digested DNA revealed considerable loss of methylation following the treatment. Cellular and histological preparations of the testis showed complete inhibition of differentiation into spermatocytic stage. Analysis of protein synthesis in the treated and control testis by growing the cells in 35S-Methionine medium and resolving the lysate by SDS-PAGE revealed that the programme of expression of at least 5 polypeptides (35.0, 31.5, 27.0, 22.5, and 18.0 KD) was altered as a result of 5-azaCdR incorporation. It appears that DNA methylation plays a critical role in the differentiation of gonia into primary spermatocytes.

Nine herbicides and pesticides were tested for their mutagenicity using the Drosophila sex-linked recessive lethal mutation assay. These are Ambush, Treflan, Blazer, Roundup, 2,4-D Amine, Crossbow, Galecron, Pramitol, and Pondmaster. All of these are in wide use at present. Unlike adult feeding and injection assays, the larvae were allowed to grow in medium with the test chemical, thereby providing long and chronic exposure to the sensitive and dividing diploid cells, i.e., mitotically active spermatogonia and sensitive spermatocytes. All chemicals induced significant numbers of mutations in at least one of the cell types tested. Some of these compounds were found to be negative in earlier studies. An explanation for the difference in results is provided. It is probable that different germ cell stages and treatment regimens are suitable for different types of chemicals. larval treatment may still be valuable and can complement adult treatment in environmental mutagen testing.

Seventy chemicals were tested for the ability to induce sex-linked recessive lethal (SLRL) mutations in postmeiotic and meiotic germ cells of male Drosophila melanogaster. As in the previous studies in this series, adult feeding was chosen as the first route of administration. If the compound failed to induce mutations by this route, injection exposure was used. Two chemicals, n-butane and propylene, were gaseous and therefore tested only by inhalation. One chemical (dimethylcarbamoyl chloride) was tested only by injection. Those chemicals that were mutagenic in the SLRL assay were further tested for the ability to induce reciprocal translocations. Sixteen of the 70 chemicals tested were mutagenic in the SLRL assay: 3-chloro-2-methylpropene, 3-(chloromethyl)pyridine HCl, dimethylcarbamoyl chloride, HC blue 1,3-iodo-1,2-propanediol, malaoxon, N,N'-methylene-bis-acrylamide, 4,4'-methylenedianiline 2HCl, ziram, cis-dichlorodiaminoplatinum II, 1,2-dibromoethane, dibromomannitol, 1,2-epoxypropane, glycidol, myleran, and toluene diisocyanate. The last seven also induced reciprocal translocations. A comparison of the results from the SLRL assay with other assays for mutagens and carcinogens suggests that the SLRL assay is highly specific, but poorly sensitive, both for mutagens and potential carcinogens.

The former U.S. EPA OPPT tiered test scheme for heritable gene mutations included the Drosophila sex-linked recessive lethal (SLRL) test in which positive results triggered the mouse specific locus (MSL) test. However, review of available literature indicated that the evaluation of mutations in the germ cells of this insect is not a good predictor of the risk of heritable gene mutations in mammals. The database contained 29 compounds for which there were conclusive MSL test results in either spermatogonial and/or postspermatogonial cells. Results in the SLRL test were available for 27 of those compounds. Of the 24 SLRL-positive chemicals, only 13 (54%) induced heritable mutations in mice; the three SLRL-negative compounds were nonmutagenic in mouse germ cells. The overall concordance between the two tests was 59%. In contrast, results of unscheduled DNA synthesis (UDS: 18 chemicals) and alkaline elution (AE: 14 chemicals) assays in rodent testicular cells following in vivo exposure correlated well with results in the MSL test (83% and 86%, respectively). MSL test results in spermatogonia and postspermatogonia were also compared separately to the SLRL, UDS, and AE assays. The concordances for the two cell types in the SLRL relative to the MSL test were 36% and 79%, respectively, indicating that the SLRL test is extremely poor in predicting heritable gene mutations in mammalian spermatogonia. Concordances for UDS and AE assays relative to MSL test results in spermatogonia (53% and 54%, respectively) and postspermatogonia (91% and 100%, respectively) were greater. Based on these analyses, the U.S. EPA OPPT has revised its tiered test scheme using assays for interaction with gonadal DNA (e.g., UDS and AE) in place of the SLRL test.

A micronucleus assay based on cytogenetic analysis of early spermatids (Tates et al.; Mutation Research 121:131-138, 1983) was applied to determine if the chelating agent ethylenedinitrilotetraacetic acid (EDTA) may induce aneuploidy in mouse meiotic cells. Previous results indicated aneuploidogenic activity of EDTA in Drosophila female germ cells (induction of chromosome loss; Zordan et al.: Environ Mol Mutagen 15:205-213, 1990). In the same study, a standard aneuploidy test based on chromosome counting in mouse secondary spermatocytes failed however to show aneuploidogenic properties of EDTA in mouse somatic and germ cells. In the present study the effects of two clastogens, adriamycin (ADM) and mitomycin C (MMC), and of the aneuploidogenic agent chloral hydrate (CH) were also evaluated. All compounds were tested at a single dose level and at two time intervals corresponding to the treatment of diakinesis/metaphase I/metaphase II spermatocytes. The clastogenic potential of the compounds under study was also evaluated, by chromosomal aberration analysis in mouse spermatogonia, in an independent set of experiments. The results obtained indicate that ADM, CH and EDTA are able to induce micronuclei at meiosis. On the contrary, only ADM and MMC induced chromosomal aberrations in mouse spermatogonia. Therefore, the most probable origin of micronuclei produced by CH and EDTA is whole chromosome lagging. These results provide further evidence for the aneuploidogenic properties of these chelating agents.

Eleven chemicals for which there has been considerable human exposure were chosen for study by the mouse specific-locus method because of their positive mutagenic action in other test systems. All were positive in the Drosophila sex-linked recessive lethal test, and all of the ten tested in mammalian somatic cells proved mutagenic. Positive results were also obtained in other tests. In contrast, in mouse stem-cell spermatogonia none of the chemicals, even at maximum tolerated dose, has given a specific-locus mutation frequency higher than the control, and the mutation frequency for all eleven combined (12 mutations in 298, 502 offspring) is actually less than the historical control, though not, of course, significantly lower. Absence of mutation induction cannot be attributed to: failure of the chemicals to reach the testis (10 of them are known to reach the testis in active form), small sample size (the samples are large), insensitivity of the test (the test is not insensitive: a positive control gave a mutation frequency 132 times higher than the historical control). It is concluded that mammalian stem-cell spermatogonia have an effective repair capability. This is supported by the dose-response and dose-fractionation results with ethyl-nitrosourea. Therefore, positive genetic hazard predictions from short-term tests on chemicals may frequently give false warning of what to expect in mammalian spermatogonia, the cells considered to be of major concern for genetic risk in human males.

The herbicide 2,4,5-T (2,4,5-trichlorophenoxyacetic acid) was evaluated for potential mutagenicity by a Salmonella/mammalian-microsome test, a dominant lethal test on female rats, and by a cytogenetic assay on spermatogonia of Chinese hamster. In the Salmonella/mammalian-microsome test on four Salmonella typhimurium strains (TA 1535, TA 100, TA 1537, and TA 98), doses of up to and including 2500 micrograms/plate did not cause any mutagenic effects. In a dominant lethal test on female rats, 8-week dietary administration of 2,4,5-T at doses of up to and including 10 mg/kg/day did not cause any increase in preimplantation loss or the rate of dead implants, and did not have any effect on the fertilization quota. Cytogenetic analysis of the spermatogonia of male Chinese hamsters orally dosed five times at 24-hr intervals with 2,4,5-T at levels of up to and including 100 mg/kg did not provide any indication of 2,4,5-T having chromosome-damaging effects. Therefore, none of the three test systems provided any indication of 2,4,5-T having a mutagenic effect.