Antimutagenic effects on male germ cells of mice

The alkylating agent cyclophosphamide (CPA) and the antioxidant ethoxyquin (EQ) were administered perorally to NMRI mice. The strong clastogenic action of CPA on spermatogonia was diminished by simultaneous doses of EQ. Higher doses of the antioxidant produced greater anticlastogenic action. Furthermore, the action of the mutagen and the antioxidant on the late spermatids and the spermatozoa was observed using the dominant lethal test. The antioxidant had only a weak influence on these postmeiotic stages.     

Cytogenetic effects of influenza virus infection on male germ cells of mice

Summary Swiss albino male mice were administered two doses (1 and 2 HA units) of influenza A₂ Hong Kong/68 virus IP. The incidence of chromosomal anomalies in spermatocytes was analysed at various times post infection and was found to be significantly higher than in controls, indicating that the influenza virus had induced these anomalies.This report is part of the PhD thesis of this author     

Cytotoxic and genotoxic effects of methotrexate in germ cells of male Swiss mice

Methotrexate (MTX) is an anti-metabolite drug widely used in the treatment of neoplastic disorders, rheumatoid arthritis and psoriasis. Developed as an analogue of folic acid, it inhibits purine and pyrimidine synthesis that accounts for its therapeutic efficacy as well as for its toxicities. MTX has narrow therapeutic index and its toxicity has been reported in various organ systems including gastrointestinal, haematologic and central nervous system. The objective of the present study is to investigate the germ cell toxicity induced by MTX in male Swiss mice. MTX was administered intraperitoneally (ip) at the doses of 5, 10, 20 and 40mg/kg to mice (20-25g) weekly once (wk) for 5 and 10 weeks. The animals were sacrificed 1 week after receiving the last treatment of MTX. The germ cell toxicity was evaluated using testes weight (wt), sperm count, sperm head morphology, sperm comet assay, histology, TUNEL and halo assay in testis. MTX treatment significantly reduced the sperm count and increased the occurrence of sperm head abnormalities in a dose dependent manner. It induced the testicular toxicity as evident from the histology of testis. Sperm comet, TUNEL and halo assay in testis also revealed significant DNA damage after MTX treatment. On the basis of the present study, it can be concluded that MTX induced germ cell toxicity in mice.     

Aging of male germ line stem cells in mice

In the present study, we investigated the effect of aging on spermatogonial stem cells (SSCs) and on the testicular somatic environment in ROSA26 mice. First, we examined testis weights at 2 mo, 6 mo, 1 yr, and 2 yr of age. At 1 and 2 yr, bilateral atrophied testes were observed in 50% and 75% of the mice, respectively; the rest of the mice had testis weights similar to those of young mice. Next, we evaluated the number and the activity of aged SSCs using spermatogonial transplantation. Numbers of SSCs in atrophied testes decreased in an age-dependent manner to as low as 1/60 of those in testes of young mice. Numbers of SSCs in nonregressed testes were similar regardless of age. The colony length, which is indicative of the potential of SSCs to regenerate spermatogenesis, was similar with donor cells from atrophied testes of 1-yr-old mice and those from testes of young mice, suggesting that SSCs remaining in 1-yr atrophied testes were functionally intact. Colonies arising from SSCs derived from 2-yr atrophied testes were significantly shorter, however, indicating that both SSC numbers and activity declined with age. Finally, we transplanted donor cells from young animals into 1- and 2-yr atrophied testes. Although the weight of 2-yr testes did not change after transplantation, that of 1-yr testes increased significantly, indicating that 1-yr, but not 2-yr, atrophied testes are permissive for regeneration of spermatogenesis by SSCs from young mouse testes. These results demonstrate that both SSCs and somatic environment in the testis are involved in the aging process.     

Cytogenetic effects of replicating and nonreplicating strains of influenza virus on male germ cells of mice

Summary Evidence is presented to demonstrate that replicating (A₂ and B) and nonreplicating (C) strains of influenza virus were capable of inducing higher percentages of chromosome anomalies in spermatocytes of mice inoculated either IP or IN than were seen in controls.This report is part of the Ph. D. thesis of this author     

Synergistic effects of germ cell expressed genes on male fertility in mice

Over 200 genes have been shown to be associated with infertility in mouse models. However, knockout mice reveal unexpected functional redundancy of some germ cell expressed genes. Single null mutations in mouse genes encoding four male germ cell proteins, transition protein 2 (Tnp2), proacrosin (Acr), histone H1.1 (H1.1), histone H1t (H1t) and sperm mitochondria-associated cysteine-rich protein (Smcp) have been generated and analysed. Tnp2 is believed to participate in the removal of the nuclear histones and initial condensation of the spermatid nucleus. Proacrosin is an acrosomal protease synthesized as a proenzyme and activated into acrosin during the acrosome reaction. The linker histone subtype H1.1 belongs to the group of main-type histones and is synthesized in somatic tissues as well as in germ cells during the S-phase of the cell cycle. The histone gene Hist1h1t is expressed exclusively in spermatocytes and may have a function in establishing an open chromatin structure for the replacement of histones by transition proteins and protamines. Sperm mitochondria-associated cysteine-rich protein (Smcp) is a major structural element of the mitochondria in the midpiece of the sperm tail. Male mutant mice lacking any of these proteins show no apparent defects in spermatogenesis or fertility. To examine the synergistic effects of these proteins in spermatogenesis and during fertilization four lines of double knockout mice Hist1h1a/Mcsp, Hist1h1t/Mcsp, Tnp2/Mcsp and Acr/Mcsp were established. It was found that even when knockout mice are heterozygous for one allele (-/+) and homozygous for the other allele (-/-), mice were subfertile. Homozygous double knockout mice of all four lines are nearly infertile. However, in the four homozygous double knockout mouse lines, different characteristic abnormalities are prominently manifested: In Hist1h1a-/-/Mcsp-/- the migration of spermatozoa is disturbed in female genital tract, in Hist1h1t-/-/Mcsp-/- spermatozoa show morphological head abnormalities, in Tnp2-/-/Mcsp-/- the motility of sperm is affected, and in Acr-/-/Mcsp-/- the sperm-oocyte interaction is impaired. These findings indicate strongly that male germ cell expressed genes have synergistic effects on male fertility.     

Reciprocal translocations in germ cells of male mice receiving external gamma-irradiation

Data reported in the literature up to 1985 on reciprocal translocation induction in male mouse germ cells by external gamma-ray doses ranging from 0.5 to 6.0 Gy delivered at fixed dose rates were analyzed. On the assumption of a non-threshold linear dose response, zero effect at zero dose, and a center of distribution lying on an approximately straight line, calculations were made of linear regression coefficients. These coefficients (b), as a function of the dose rate (P), were well fitted by two straight lines: b = (3.15 +/- 0.59 log P) X 10(-6) for dose rates from 0.01 to 0.1 mGy/min; and b = (7.52 +/- 3.86 log P) X 10(-6) for dose rates ranging from 0.06 to 1.2 X 10(3) mGy/min. The intersection point of these two lines determined the so-called threshold level of the dose rate, namely, 4.6 X 10(-2) mGy/min, at which the effectiveness of external gamma-irradiation is not expected to exceed 2.36 X 10(-6)/mGy. In addition, experiments were undertaken in which yields were recorded of reciprocal translocations in germ cells of male mice exposed to 0.9 Gy of gamma-radiation at dose rates ranging from 6.14 X 10(-3) to 6.14 X 10(2) mGy/min (6 levels); comparisons were made with data published up to 1985 from similar studies using other fixed doses. To do this, translocation yields were expressed as relative yields (F) and their relationship to the dose rate (P) for the individual fixed doses was represented by an equation of the type: F = alpha + beta log P. For most of the equations, the regression coefficients were in good agreement and a single relationship was obtained to represent them. From the analysis performed it follows that, within the 0.6-6.0 Gy dose range, the pattern of the F vs. P relationship is unaffected by the dose. This supports the initial assumption that for the dose range up to 6.0 Gy the dose response for the reciprocal translocation yield is a non-threshold straight-line relationship.     

Identification and developmental expression of a smooth-muscle gamma-actin in postmeiotic male germ cells of mice.

Mouse testis contains two size classes of actin mRNAs of 2.1 and 1.5 kilobases (kb). The 2.1-kb actin mRNA codes for cytoplasmic beta- and gamma-actin and is found throughout spermatogenesis, while the 1.5-kb actin mRNA is first detected in postmeiotic cells. Here we identify the testicular postmeiotic actin encoded by the 1.5-kb mRNA as a smooth-muscle gamma-actin (SMGA) and present its cDNA sequence. The amino acid sequence deduced from the postmeiotic actin cDNA sequence was nearly identical to that of a chicken gizzard SMGA, with one amino acid replacement at amino acid 359, where glutamine was substituted for proline. The nucleotide sequence of the untranslated region of the SMGA differed substantially from those of other isotypes of mammalian actins. By using the 3' untranslated region of the testicular SMGA, a highly specific probe was obtained. The 1.5-kb mRNA was detected in RNA from mouse aorta, small intestine, and uterus, but not in RNA isolated from mouse brain, heart, and spleen. Testicular SMGA mRNA was first detected and increased substantially in amount during spermiogenesis in the germ cells, in contrast to the decrease of the cytoplasmic beta- and gamma-actin mRNAs towards the end of spermatogenesis. Testicular SMGA mRNA was present in the polysome fractions, indicating that it was translated. These studies demonstrate the existence of an SMGA in male haploid germ cells. The implications of the existence of an SMGA in male germ cells are discussed.     

Differentiation of mouse primordial germ cells into female or male germ cells.

Mouse primordial germ cells (PGCs) migrate from the base of the allantois to the genital ridge. They proliferate both during migration and after their arrival, until initiation of the sex-differentiation of fetal gonads. Then, PGCs enter into the prophase of the first meiotic division in the ovary to become oocytes, while those in the testis become mitotically arrested to become prospermatogonia. Growth regulation of mouse PGCs has been studied by culturing them on feeder cells. They show a limited period of proliferation in vitro and go into growth arrest, which is in good correlation with their developmental changes in vivo. However, in the presence of multiple growth signals, PGCs can restart rapid proliferation and transform into pluripotent embryonic germ (EG) cells. Observation of ectopic germ cells and studies of reaggregate cultures suggested that both male and female PGCs show cell-autonomous entry into meiosis and differentiation into oocytes if they were set apart from the male gonadal environments. Recently, we developed a two-dimensional dispersed culture system in which we can examine transition from the mitotic PGCs into the leptotene stage of the first meiotic division. Such entry into meiosis seems to be programmed in PGCs before reaching the genital ridges and unless it is inhibited by putative signals from the testicular somatic cells.     

Genetic effects of combined chemical-X-ray treatments in male mouse germ cells

Several studies have shown that the yield of genetic damage induced by radiation in male mouse germ cells can be modified by chemical treatments. Pre-treatments with radio-protecting agents have given contradictory results but this appears to be largely attributable to the different germ cell stages tested and dependent upon the level of radiation damage induced. Pre-treatments which enhance the yield of genetic damage have been reported although, as yet, no tests have been conducted with radio-sensitizers. Another form of interaction between chemicals and radiation is specifically found with spermatogonial stem cells. Chemicals that kill cells can, by population depletion, substantially and predictably modify the genetic response to subsequent radiation exposure over a period of several days, or even weeks. Enhancement and reduction in the genetic yield can be attained, dependent upon the interval between treatments, with the modification also varying with the type of genetic damage scored. Post-treatment with one chemical has been shown to reduce the genetic response to radiation exposure.     

Transplantation of male germ line stem cells restores fertility in infertile mice

Azoospermia or oligozoospermia due to disruption of spermatogenesis are common causes of human male infertility. We used the technique of spermatogonial transplantation in two infertile mouse strains, Steel (Sl) and dominant white spotting (W), to determine if stem cells from an infertile male were capable of generating spermatogenesis. Transplantation of germ cells from infertile Sl/Sld mutant male mice to infertile W/Wv or Wv/W54 mutant male mice restored fertility to the recipient mice. Thus, transplantation of spermatogonial stem cells from an infertile donor to a permissive testicular environment can restore fertility and result in progeny with the genetic makeup of the infertile donor male.     

Cytogenetic effects of microwave irradiation on male germ cells of the mouse

Hybrid male mice were exposed to 2.45 GHz microwaves for 30 min/day, 6 days a week for two consecutive weeks at power densities of 1.0, 100 or 400 W m-2, with sham-exposed controls. Rectal temperatures before and after exposure were measured on days 1, 6 and 12. Measurements made on day 1 were treated with caution because of heterogeneity in rectal temperatures taken before exposure between the groups of mice given different treatments. On days 6 and 12, rectal temperatures rose by approximately 1 degree C in mice sham exposed, or exposed to 1 W m-2 or 100 W m-2. Only in the group of mice exposed to 400 W m-2 was the mean rise in rectal temperature during exposure (about 3 degrees C) significantly increased above the sham value. In groups killed 2-3 days after treatment (mainly meiotic exposure) frequencies of chromosome aberrations in spermatocytes showed no significant heterogeneity although the highest frequency of 1.5 per cent was at the highest (400 W m-2) power density. Another group killed 30 days after 100 W m-2 exposures (spermatogonial sampling) showed no significant increase over controls in chromosome aberration frequency. There was a small but significant increase in sperm count with increasing power density in mice killed 12-13 days after exposure, but a non-significant one in those exposed as spermatogonia (killed 41 days later). Thus effects were markedly less severe than those reported previously by Manikowska-Czerska et al. (1985) with a very similar radiation regime and were probably caused by the temperature enhancement.     

Cytogenetic effects of malathion insecticide on somatic and germ cells of mice

Male mice dermally exposed to single or multiple treatment (5 days/2 weeks) showed that the ability of malathion to induce chromosome aberrations in somatic (bone marrow) and germ cells (primary spermatocytes) was related to the type of treatment and dose used. Statistically significant increases of chromosome aberrations in bone marrow cells occurred after single treatment (500 and 2000 mg/kg body wt) when chromatid gaps were included and after multiple treatment (250 and 500 mg/kg) when they were excluded. No dose-response relationships were observed for either treatment. In germ cells, malathion induced a significant increase of univalents in both types of treatment but structural chromosome aberrations were induced only by multiple treatment. Malathion induced a significant decrease of the mitotic indices in the bone marrow.