Synaptonemal complex analysis of X-7 translocations in male mice

The synaptonemal complexes of surface-spread spermatocytes of mice heterozygous for one of two reciprocal translations (R3 and R5) between the X and chromosome 7 have been examined by light and electron microscopy (EM). The break points of R3 were determined to be at 70% of chromosome 7, as measured from the centromere, and at 22% of the X. Translocation quadrivalents were formed almost exclusively. The break points of R5 were at 21% of chromosome 7 as measured from the centromere, and at 83% of the X. There was little indication that the break in the X interfered with sex-chromosome synapsis between the 7X and Y. Univalent Y's were not observed in R3, and only seldom observed (8–14%) in R5. However, in contrast to R3, R5 formed quadrivalents relatively rarely (20% in the EM study of 100 nuclei), and heteromorphic bivalents of 7X-Y and X7-7 quite frequently (72%). Possible causes of this high bivalent frequency are discussed. Light-microscope (LM) analysis alone was found to be inadequate for interpreting synaptic configurations (quadrivalents vs. bivalents) in R5. The LM analysis was further complicated by the occurrence of nonhomologous synapsis in the heteromorphic bivalents of R5, a phenomenon easily recognized and interpreted in the EM portion of the study.     

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.     

Cytogenetic and molecular analysis of male infertility

Reduced male fertility and subfertility can be caused by genetic factors that affect both germ cell development, differentiation, and function; in particular, chromosome abnormalities and Yq microdeletions are a possible cause of spermatogenetic impairment in males as shown by their higher frequency in infertile men than in the general male population. Microdeletion of the long arm of the Y chromosome (Yq) are associated with spermatogenic failure and have been used to define three regions on Yq (AZFa, AZFb, and AZFc) that are critical for germ cell development. With the advent of assisted reproductive technology and intracytoplasmic sperm injection, knowledge about the various factors leading to spermatogenic impairment is one of the most important aspects of scientific research. Therefore, this study was designed to identify the frequency of cytogenetic and submicroscopic interstitial deletions in azoospermia factor loci in infertile Indian males. One hundred and eighty males with nonobstructive oligozoospermia and azoospermia were included in this study. Semen analysis was done in each case to determine the spermatogenic status. Individuals were subjected to detailed clinical examination, family history, and endocrinological and cytogenetic study after consent from the patient. Peripheral blood cultures were set up according to standard protocols and 30 G-banded metaphases were analyzed in each case. Numerical and structural chromosomal abnormalities were detected in 40 infertile cases. Fluorescence in situ hybridization analysis was done in some cases to identify the percentage of mosaic cell lines and any cryptic or low-level mosaicism. Polymerase chain reaction microdeletion analysis was done in 140 cytogenetically normal cases. Of the 140 cases, 8 showed deletion of at least one of the sequence-tagged site markers. Review of literature has shown that the overall frequency of microdeletions varies from 1 to 55%. In the present study, the frequency of microdeletion was 5.8%, and deletions were identified in cases with undescended testis and varicocele and cases with bilateral severe testiculopathy.     

Strategies for detecting heritable translocations in male mice by fertility testing

Data from a heritable translocation test were analysed to estimate the best rule for classification of F₁ males in normals or partially sterile translocation carriers according to litter size or numbers of live and dead implants per mating. Six rules were com[ared for classification with up to three litter sizes per F₁ male observed. The results indicate that a translocation rate of 2%, or at best of 1%, can be detected with reasonable cost.     

Molecular analysis of gene conversion in spermatids from transgenic mice

Investigations into the mechanisms and properties of gene conversion in mammals are greatly restricted by the inability to recover all the products of a meiosis. Additionally, the study of this process has been hampered by the lack of visible markers to detect gene conversion, especially when the events are rare. In previous work, we developed a transgenic system for detection and quantitation of gene conversion events in the germline of mice (Murti, J.R., Bumbulis, M., Schimenti, J.C., 1992. High frequency germline gene conversion in transgenic mice. Mol. Cell. Biol. 12, 2545–2552) that could be exploited as an assay for recombinogenic chemicals (Murti, J.R, Schimenti, K.J., Schimenti, J.C., 1994. A recombination-based transgenic mouse system for genotoxicity testing. Mutat. Res. 307, 583–595). A specific intrachromosomal gene conversion event between two complementarily defective lacZ genes resulted in the production of β-galactosidase in spermatids, enabling a measurement of conversion frequency. Here, we report that the anticancer drug, cisplatin, increased gene conversion in meiotic stage cells in these transgenic mice. Furthermore, a method was developed for direct molecular analysis of transgene conversion events in single or pooled lacZ-positive spermatids. The ability to identify gametes that have undergone a rare gene conversion event, followed by molecular amplification of the recombinant gene, should make it possible to investigate the mechanisms of genetic recombination in mammals in greater detail than previously possible.     

Synaptonemal complex analysis of X-7 translocations in male mice: R2 and R6 quadrivalents

Synaptonemal complexes of surface-spread spermatocytes of mice heterozygous for reciprocal translocations R2 or R6 between the X-chromosome and chromosome 7 were examined by light and electron microscopy (EM). Measurements of the lengths of all chromosome axes involved in the translocation configurations and of the extent of synapsis were used to calculate the position of the break points of the two translocations. The breaks for R2 were determined to be at 62% of the 7 as measured from the centromere, and at 27% of the X. Quadrivalents were formed almost exclusively. The break points for R6 were calculated to be at 30% of the 7 as measured from the centromere, and at 75% of the X. Although in R6 the break in the X lies within the potential pairing region of the sex chromosomes, univalent Ys were rarely observed (6%). The EM sample of 76 nuclei contained: 42% quadrivalents, 52% heteromorphic bivalents, 4% trivalent plus Y univalent, and 2% X7-7 bivalent plus two univalents (7X and Y). Nonhomologous synapsis occurred in the quadrivalents of both R2 and R6. In R6 nonhomologous synapsis of the X portion of the 7X with the 7 involved up to 14% of the length of the 7. Methods are discussed for determining the position of the break points in the presence of nonhomologous synapsis. It is proposed that the high percentage of bivalents is due to premature desynapsis of the 7X from the 7 and that the X portion of the 7X axis confers its property of premature desynapsis on that portion of the 7 to which it is attached.     

Heritable translocations induced by dermal exposure of male mice to acrylamide

Acrylamide (AA) is an important industrial chemical used mainly in the production of polymers. It can be absorbed through the skin. AA was shown to be a germ cell clastogen that entails a genetic risk for exposed workers. The genetic risk calculation was based on mouse heritable translocation test data obtained after acute intraperitoneal (ip) exposure (Adler et al., 1994). To obtain a correction factor between ip and dermal exposure, dominant lethal and heritable translocation tests were carried out with dermal exposure of male mice to AA. In the dominant lethal test, male (102/El x C3H/El)F1 mice were exposed by dermal application to the shaved backs of 50 mg/kg AA per day on five consecutive days or to five daily ip injections of 50 mg/kg AA. One day after the end of exposure, the males were mated to untreated females of the same hybrid stock for four days and females were changed every four days for a total of five matings. Dominant lethal effects were found during matings 1-3. For ip exposure, these values were 81.7, 85.7 and 45.4%, respectively; for dermal exposure the corresponding values were 22.1, 30.6 and 16.5%, respectively. In the heritable translocation assay, male C3H/El mice were treated with five dermal exposures of 50 mg/kg AA and mated 1.5-8.5 days after the end of exposure to untreated female 102/El mice. Pregnant females were allowed to come to term and all offspring were raised to maturity. Translocation carriers among the F1 progeny were selected by a sequential fertility testing and cytogenetic analysis including G-band karyotyping and M-FISH. A total of 475 offspring were screened and 41 translocation carriers were identified. The observed translocation frequency after dermal exposure was 8.6% as compared to 21.9% after similar ip exposure (Adler, 1990). The calculated ratio of ip vs. dermal exposure of 0.39 can be applied to obtain a more realistic calculation of genetic risk for dermally exposed workers.     

Strategies for detecting heritable translocations in male mice by fertility testing.

Data from a heritable translocation test were analysed to estimate the best rule for classification of F1 males in normals or partially sterile translocation carriers according to litter size or numbers of live and dead implants per mating. Six rules were compared for classification with up to three litter sizes per F1 male observed. The results indicate that a translocation rate of 2%, or at best of 1%, can be detected with reasonable cost.     

Cytogenetic effects of myleran in vivo on bone-marrow cells from male mice

The cytogenetic effects of Myleran were examined in bone-marrow cells from male mice. To study the dose-response relationship the male mice were injected with 5, 10, 20 or 40 mg of Myleran/kg. Bone-marrow samples were prepared 24 h later. The time response was investigated by examining bone-marrow samples 1, 2, 4 or 10 days after i.p. injection of 40 mg of Myleran. Most of the structural aberrations were of the chromatid type and the dose-response relationship was linear. The chromatid and chromosomal aberrations were maximal at 2 days and decreased sharply after longer intervals of time.     

Cytogenetic effects of protracted gamma exposures from conception of male mice

In order to gain an overall picture of the genetic effects of an increased level of background radiation it is necessary to study the results of protracted exposures to embryonic and immature germ-cell stages as well as to stages found in the mature organism. For this purpose, litters produced by female mice, kept in a 10 or 20 rad/day ⁶⁰Co-γ-irradiation field, were kept in the same fields from conception until about 60 days later, having absorbed doses of 526 and 1078 rad respectively. Tests on exposed female offspring showed them to be sterile. 8 weeks after removal from the gamma field, mean testis masses of males in the 20 rad/day series were only half normal but those receiving 10 rad/day were little affected. Frequencies of translocations in spermatocytes at diakinesis/metaphase I were only slightly increased in the exposed series, differences not being significant. Estimated rates of translocation induction were around 5 × 10^?6 per rad, about one-third of those found after protracted γ-irradiation of stem-cell spermatogonia in the adult. Embryonic lethality in progeny of other similarly irradiated males (absorbed doses of 560 and 1040 rad), mated 2 months after removal from the radiation fields, was also increased slightly, but not significantly. Results are compared with others on the induction of chromosome aberrations and gene mutations, mainly by acute irradiation, in prenatal and neonatal male mice. It is concluded that early male germ-cell stages generally show a reduced genetic radiosensitivity after both acute and chronic exposures.     

Transmission of X-ray-induced reciprocal translocations in normal male mice and in male mice with a reduced sperm count due to translocation homozygosity

Normal (+/+) and translocation T(1; 11.13S)70H homozygous (T/T) male mice received 2 X 2.5 Gy X-rays with a 24-h interval. After 120 days, the frequency of late diplotene-metaphase I spermatocytes with translocation multivalents was 14.1% for +/+ and 13.7% for T/T males, respectively, in one group of animals of each type. The difference is not significant. A second group was allowed to sire progeny for 60 days with 2 normal females per week. Reciprocal translocations detectable at diakinesis/metaphase I were observed in 2.5% of the 395 male progeny from the irradiated +/+ fathers, and in 2.9% of the 489 male progeny from the irradiated T/T fathers. This leads to a pooled estimated transmission of 0.81 +/- 0.19. Translocations induced in the long 11.13 metacentric chromosome were not transmitted with a different frequency. The rate of heritable induced translocations in this study was 5.4 X 10(-5)/rad/gamete. On the basis of the data of Generoso et al. (1984) for the frequency of the heritable spontaneous translocations in male mice, it is concluded that, because of their low doubling dose (3.3-4.6 rad), the spontaneous translocations are probably of postmeiotic origin.     

X-Autosome translocations: Cytogenetic characteristics and their consequences

Summary To define the principal characteristics of X-autosome translocations, the authors present a study of 105 cases, five of which are personal observations. The autosomal pairs 15, 21, and 22 are affected by t(X-Aut) more often than would be expected. The distribution of breakpoints on the X chromosome does not differ significantly from the expected distribution. The analysis of different patterns of inactivation seems to confirm that the inactivation could occur at random, but would be followed by a cellular selection favoring the better genetic balance. An estimate of the incidence of t(X-Aut) is proposed, based upon the conclusions that only one chromosome is susceptible to translocation in meiosis in both males and females and that all affected men will be sterile, as will be 50% of women.     

Unequal nondisjunction frequencies of trivalent chromosomes in male mice heterozygous for two Robertsonian translocations

Robertsonian (Rb) translocation heterozygosity may cause pairing problems during prophase and segregation irregularities at anaphase of meiosis I. These stages of meiosis I were studied in male mice doubly heterozygous for the two Rb chromosomes Rb(9.19)163H and Rb(16.17)8Lub. At pachytene both Rb chromosomes similarly showed pairing irregularities like unpaired segments. However, highly different nondisjunction frequencies of chromosomes forming the respective trivalents were found. The nondisjunction frequency of the Rb8Lub trivalent chromosomes was about 40%, whereas a very low frequency of nondisjunction was found in combination with the Rb163H trivalent. Since both trivalents were together in the same cell, differences in kinetochore function are assumed to be responsible for the diverse frequency of nondisjunction.     

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     

Cytogenetic effect of 238Pu alpha radiation on the germ cells of male mice

A study was made of the yield of reciprocal translocations in stem spermatogonia of mice exposed to alpha-radiation (238Pu) and whole-body acute and chronic gamma-radiation within a wide range of doses and dose-rates. The frequency of reciprocal translocations induced by a single intraperitoneal administration of plutonium nitrate was relatively low and independent of the dose 1.5-18 months after the effect. The yield of the reciprocal translocations induced by chronic effect of gamma-radiation was appreciably lower than that observed after acute irradiation with the same dose and grew linearly with dose. The RBE of alpha-radiation was 10-20 with respect to chronic effect of gamma-radiation.     

Cytogenetic investigation of meiotic chromosomes of male mice after chronic caffeine treatment

Summary Male C3H inbred mice and male (101xC3H) F₁ hybrids were treated with various caffeine doses over a long period. After the end of treatment the animals were dissected and the metaphase I figures of the cycle of spermatogenesis were analysed. The experiments revealed no distinct evidence for the induction of chromosomal aberrations in the cycle of spermatogenesis caused by caffeine up to metaphase I.This study was supported by the Deutsche Forschungsgemeinschaft.     

Distribution of chiasmas in the 2d and 6th chromosomes involved in Robertsonian translocations in male mice

The distribution of chiasmata in 2 and 6 chromosomes in males homozygous for Rb(2.6)4Iem and Rb(8.17)1Iem was studied. Chiasmata were shown to distribute along chromosomes non-randomly, exchanges occurring in telomeric regions. Chiasmata distribution is substantially different for the cases of one and two chiasmata per bivalent. The main cause for these differences is supposed to be strong positive chiasmata interference (the position of the first chiasma may determine the position of the second one). The centromere blocks this effect, so chiasma in one arm does not interfere with that in the second arm. It has been shown that the frequency of double exchanges depended on not only the distance between markers under study, but also on marker position in the chromosome.