E.P. Evans

An Air-Drying Method for Meiotic Preparations from Mammalian Testes

A suspension is made in isotonic (2.2%) sodium citrate solution from the contents of the tubules from a whole testis or a testicular biopsy specimen. The germinal cells are sedimented by centrifuging, leaving most of the sperm in the supernatant fluid, which is discarded. The cells are resuspended in hypo-tonic (1%) sodium citrate solution and left to stand at room temperature for 12 minutes, after which they are sedimented again and fixed as a concentrated suspension in a mixture of 3 parts absolute ethyl alcohol to 1 part glacial acetic acid plus a trace of chloroform. Two quick changes into fresh fixative follow. Air-dried preparations are made from the final fixed suspension and stained in lactic-acetic-orcein. The method is suitable for stages of male meiosis in which the chromosomes are condensed. Its principle advantage is the separation of the clumps of spermatogonia and spermatocytes into individual cells which are randomly dispersed over the preparations. Compared with squash techniques, the air-drying method gives improved spreading of the chromosomes and less cell breakage.

Differential transmission of translocations induced in spermatogonia of mice by irradiation

The hindquarters of two batches each of five young adult male mice were exposed to an acute dose of 1,200 R of 250-kV X-rays in two fractions of 600 R, separated by a period of eight weeks. Means of 4

Cytogenetic and endocrine studies of a freemartin heifer and its bull co-twin

The study of a bovine freemartin and its male co-twin has shown that the sex-reversed gonads of the female are secreting testosterone. Therefore, we suggest that the masculinisation of the freemartin’

Studies on the induction of translocations in mouse spermatogonia II. Effects of fast neutron irradiation

Abstract The rate of induction of reciprocal translocations by fast neutron irradiation of mouse spermatogonia was studied by cytological examination of descendant spermatocytes. With acute irradiation (49–55 rad/min) the frequency of affected spermatocytes rose to a maximun of 8.7% at 100 rad, falling abruptly at higher doses to 1.6% at 220 rad. With chronic irradiation over 12 weeks, however, no such fall-off was found. Instead, the yield at 214-rad neutrons was 21.7%, while that at 62-rad was 3.3%, which is significantly less than expected on a linear hypothesis. If one assumes linearity at low doses, the neutron-X RBE for acute irradiation is about 4 and the neutron-γ RBE for chronic irradiation is about 20–25.

Studies on the induction of translocations in mouse spermatogonia. Iii. Effects of x-irradiation.

Abstract Translocation frequencies were studied in mouse spermatocytes derived from X-irradiated spermatogonia. Over the range from 50–800 R the dose-response relationship did not differ significantly from linearity, in agreement with previous findings of Leonard and Deknudt . It seems probable that the initial dose-response curve has the expected square-law component but becomes destorted by secondary factors between irradiation and meiotic examination of cells. Observed translocation frequencies were little affected by increasing length of time (from 11–30 weeks) between irradiation and examination, except for a possible decline at the highest dose (800 R). No significant frequency differences were found between mated and solitary males, or between those given whole-body or part-body (gonadal) irradiation.

A reciprocal translocation in the mouse between the x chromosome and a short autosome.

Preparations were obtained from the testes of six sterile males of the T(X; ?) 16H stock. 81.7% of 694 primary spermatocytes in stages from diakinesis to first metaphase contained chain quadrivalents

Studies on the induction of translocations in mouse spermatogonia. IV. Effects of acute gamma-irradiation.

Abstract The rate of induction of reciprocal translocations by 56–816 R exposures of mouse spermatogonia to acute γ-irradiation (95 R/min) was determined by cytological examination of descendant spermatocytes. The dose-response relationship did not differ significantly from linearity and had a regression coefficient of 1.8·10 −4 per R with respect to translocations per spermatocyte. Further analysis at exposures below 816 R (considered less likely to produce distortion) showed that the quadratic regression of best fit had too small a square-law component to account for the very low frequency of translocations obtained after chronic γ-exposures in a previous experiment. The possibility is discussed that there is some extra factor, besides the diminution of the square-law component, which operates to reduce the yield after protracted exposures. The relative biological effectiveness of the γ - vs . X-irradiation for the induction of translocations in spermatogonia was estimated to be 0.62 ± 0.10.

A 39,x/41,xyy mosaic mouse.

A sterile mouse was found to be a chromosomal mosaic with two cell lines, one containing 39 chromosomes, the other 41. Both types were present in approximately equal numbers in the bone marrow, but the 41-chromosome line alone was identified in spermatogonia and spermatocytes. Evidence from chromosome morphology at mitotic metaphase and from spermatocytes at diakinesis and metaphase I identified the two cell-lines as XO and XYY respectively. The alternative possibilities of exclusion of the XO line from the seminiferous elements by chance and by selection are discussed and the latter interpretation favoured. The possibility is raised that the Y chromosome of mammals carries genetic information essential for normal spermatogenesis, as distinct from its established function in embryonic gonadal determination.

Studies on the induction of translocations in mouse spermatogonia V. Effects of short-term fractionation

Abstract Translocation frequencies were studied in spermatocytes derived from the spermatogonia of mice given 300 rad X-rays in two equal fractions separated by intervals of 0–8 h. In the first experiment (0–2 h intervals) there was a significant fall from 14.5% to 8.1% in the frequency of affected cells when there was an 0.5-h interval between doses instead of a single dose, followed by a just significant rise to 10.8% at a 2-h interval. However, the translocation frequency after a single 300-rad dose was higher than expected from previous experiments at this level (see paper III of this series). In the second experiment (0–8 h intervals) there was again an initial fall (from 8.8% at 0 h to 6.6% at 1 h) and a rise later (to 8.1% at 6 h and at 8 h intervals), but the changes in frequency did not reach a significant level. Results are compared with those obtained in similar recent experiments by Leonard and Deknudt (500 R total exposure) in which an initial decrease in yield was also found, but with more pronounced fluctuations in frequency later. The relative importance of radiation-induced changes in the spermatogonial population and of decreased oppurtunities for interaction of lesions on fractionation is discussed with reference to the observed effects.

FISH mapping of the mouse Ret oncogene to the junction of G-bands E3/F1 on Chromosome 6 indicates a need for reassessment of the physical and consensus maps

The RET proto-oncogene encodes a cell-surface glycoprotein with cytoplasmic tyrosine kinase activity (Takahashi et al., 1989). Mutations in RET have been associated with four human disease syndromes: Hirschprung’s disease, familial medullary thyroid carcinoma, and two forms of multiple endocrine neoplasia, type 2A and 2B (van Heyningen, 1994). The mouse Ret gene has been cloned (Iwamoto et al., 1993) and is expressed in normal mouse spinal cord and in Fas and Fas1 lymphadenopathy (Takahashi et al., 1988). Mice homozygous for the targeted knockout of Ret develop to term, but die soon after birth with renal agenesis/dysgenesis (Schuchardt et al., 1995). They also lack enteric neurons throughout the digestive tract. Thus the Ret gene product may be a receptor for a factor involved in development of a number of neural cell lineages, and in kidney organogenesis (Pachnis et al., 1993). Ret was assigned by interspecific backcross analysis to 53.2 cM on Chr 6, 1.1 cM distal to Raf1 (Elliott and Moore 1998, Li et al., 1995). The mapping of loci in this region on the physical map appears contradictory in that Raf1 has been positioned in G-band 6C3 by in situ hybridisation (Tailor and Martin-DeLeon, 1989), but Pparg and Sdf1, only 0.5 cM distal to Raf1 on the consensus map, have been assigned by FISH to bands E3/ F1 and F1 respectively (Zhu et al., 1995, Nomura et al., 1996). The close linkage of these loci is incompatible with these alleged G-band locations since, according to the Mouse Chromosome Atlas (Lyon and Kirby, 1996), G-bands 6C3 and F1 could be up to 10 cM apart. Del(6)Ums26H (hereafter referred to as Del(6)Ums) is a deletion mutant found by screening progeny of spermatogonially X-irradiated male mice, carriers having umbrous coats, low viability and small body size (Cattanach et al., 1993 ). The deleted chromosome segment of Chr 6 was initially thought to include all of G-band C3. If the FISH assignment of Raf1 to C3 is correct then the Ret locus may be located within the deleted region, or at either of its two breakpoints. If this is the case, mice carrying the Del(6)Ums deletion could provide potential haplo-insufficient models for the human disease syndromes associated with mutations at the RET locus. Therefore, to determine the unequivocal location of the mouse homologue within or outside the deletion Del(6)Ums, and to resolve the existing ambiguity caused by the in situ and FISH mapping of loci in this region, we have positioned Ret directly by FISH using mitotic cells from Del(6)Ums heterozygous mice, carrying both a deleted and normal Chr 6.