| First Author | Cattanach BM | Year | 1992 |
| Journal | Mouse Genome | Volume | 90 |
| Pages | 87-9 | Mgi Jnum | J:18104 |
| Mgi Id | MGI:66123 | Citation | Cattanach BM, et al. (1992) Rescue of a male sterile mutation by in vitro fertilisation. Mouse Genome 90:87-9 |
| abstractText | Full text of Mouse Genome contribution: RESCUE OF A MALE STERILE MUTATION BY IN VITRO FERTILISATION. B M Cattanach, E P Evans, C Rasberry, M Wood* and P H Glenister; MRC Radiobiology Unit, Chilton, Didcot, Oxon OX11 ORD; *St Georges Hospital Medical School, Cranmer Terrace, London SW17 ORE INTRODUCTION Morphologically abnormal animals which fail to breed are not uncommonly found in mutation experiments and, because of their infertility or incapacity to mate, evidence that their abnormalities may be due to genetic changes cannot usually be obtained. This communication describes the rescue of such a variant by a combination of in vitro fertilisation and embryo transfer techniques which has allowed its genetic basis to be distinguished. MATERIALS AND METHODS The original variant, a male, was discovered in the course of a specific locus mutation study in which spermatogonially irradiated (24h fractionated 3+3Gy X-rays) C3H/HeH x 101/H Fl (3H1) males were crossed with tester (PT) females homozygous for the recessive marker genes, non-agouti (a), brown (b), pink-eye (p), chinchilla (c(ch)), dilute (d), short-ear (se) and piebald spotting (s) and the offspring scored for mutations at these loci or for new dominant mutations at other loci. In vitro fertilisation was achieved as described by Fraser and Drury (1975). The male was killed, spermatozoa were stripped from his vasa deferentia and caudae epididymes and cultured with ova from MF1 females hormonally induced to ovulate by injection of PMS and HCG (5iu, 48hr apart). Evidence of fertilisation was sought 5h later and, after further culture until the eggs were at the 2-cell stage, these were transferred to the oviducts of host pseudopregnant B6CB Fl (C57BL/6JLac x CBA/CaLac) females. Subsequent in vitro fertilisation was carried out using spermatozoa from affected descendants of the original male; the ova were here derived from 3H1 females (except in one instance when PT females were used), and recipients were 3H1 females. RESULTS DESCRIPTION OF MUTANT The original mutant male was detected because of gross limb abnormalities in which both fore and rear limbs were shortened and distorted outwards with the consequence that the animal could move only by shuffling along on its upper limbs. The skull also appeared somewhat dome-shaped and the eyes, although of normal shape, were often gummy and seemingly liable to infection. A slight generalised tremor was also regularly noted suggesting some neurological disturbance. When placed with a series of females over several months no litters were produced and on checking the females for vaginal plugs evidence of mating was never found. Analysis of the skeletal abnormalities as visualised on X-ray plates (Fig 1) showed that the humeri of the fore limbs were fairly normal apart from the absence of the acromion processes. Both the radii and ulnae were shortened and bent (hemimelia) with the feet also reduced. The femurs of the hind limbs were similarly affected and the tibiofibulae were narrow and distorted. The metatarsals and phalanges also looked abnormal. Because of the gross distortion of the limbs the mutant was arbitrarily called limb distortion (Ldn). IN VITRO FERTILISATION AND EMBRYO TRANSFERS Motile spermatozoa were obtained from the variant male and in culture these fertilised a number of MF1 ova (Table 1). On transfer to recipient females these yielded 9 progeny, 3 of which showed the same variant Ldn phenotype as the father, so indicating a dominant gene inheritance. The yield of progeny relative to numbers of fertilised ova transferred was notably lower than that from control in vivo fertilised ova from MF1 crosses (albino) carried out concurrently using the same recipient females (Table 1). The in vitro rescue was used in two subsequent generations but eventually failed due to low fertilisation rates associated with low numbers of spermatozoa of poor quality. Overall, however, 16 Ldn and 10 wild type progeny were obtained in the rescue experiments (Table 1). Table 1 Results of Ldn rescue experiments using in vitro fertilisation Male/generation: Original; Source of ova: MF1; Ova: No. used: 151; No. 2-cell (%): 53 (35); Total born: 9; Progeny: per 2-cell: 17%; Mutant (female; male): 3 (2:1). Male/generation: Control; Source of ova: MF1; Ova: No. used: 46; No. 2-cell (%): 46; Total born: 33; Progeny: per 2-cell: 72%; Mutant (female; male): -. Male/generation: 1; Source of ova: 3H1; Ova: No. used: 222; No. 2-cell (%): 36 (16); Total born: 7; Progeny: per 2-cell: 19%; Mutant (female; male): 6 (1:5). Male/generation: 2; Source of ova: 3H1; Ova: No. used: 82; No. 2-cell (%): 42 (51); Total born: None; Progeny: per 2-cell: -; Mutant (female; male): -. Male/generation: 2; Source of ova: 3H1; Ova: No. used: 150; No. 2-cell (%): 12 (8); Total born: None; Progeny: per 2-cell: -; Mutant (female; male): -. Male/generation: 2; Source of ova: 3H1; Ova: No. used: 160; No. 2-cell (%): 24 (15); Total born: 10; Progeny: per 2-cell: 42%; Mutant (female; male): 7 (4:3). Male/generation: 3; Source of ova: PT; Ova: No. used: ?; No. 2-cell (%): None; Total born: -; Progeny: per 2-cell: -; Mutant (female; male): -. Male/generation: 3; Source of ova: 3H1; Ova: No. used: ?; No. 2-cell (%): None; Total born: -; Progeny: per 2-cell: -; Mutant (female; male): -. Male/generation: 3; Source of ova: 3H1; Ova: No. used: 250; No. 2-cell (%): 20 (8); Total born: None; Progeny: per 2-cell: -; Mutant (female; male) -. BREEDING DATA The first two mutant females produced were mated with 3H1 males and both proved capable of producing litters although their mean litter sizes were low (2.5). Of 4 others mated in later generations only one was fertile (mean litter size 3.7), the others were somewhat runted. Small size and failure to thrive became a key factor, together with the declining success rate of the in vitro rescue, in the eventual demise of the mutant. However, among 29 progeny produced from crosses of Ldn females with 3H1 males and classified at birth, 15 showed the Ldn phenotype (9 females, 6 males) and 14 were wild type (4 females, 10 males) confirming the dominant gene inheritance. CYTOGENETIC ANALYSES G-banded mitotic preparations of one Ldn female showed the presence of a reciprocal translocation between chromosomes 5 and 16, with the break- points located proximally in 5B and in 16C2. This places one breakpoint in the vicinity of reeler (rl) at the extreme proximal end of the chr 5 linkage map and the other possibly near amyloid Beta(A-4) protein (App) distally on chr16. (Lyon and Kirby, 1991). No other chromosomal abnormalities were found. The translocation, although extinct, has been designated T(5,16)53H. CONCLUSIONS While it may often be reasonably inferred that morphologically variant animals discovered following parental mutagen exposure are the result of induced mutations, the proof of this is only obtained when the characteristics are shown to be transmissible to later generations. In the case of sterile animals such evidence is usually lacking. However, the phenotypic abnormalities recorded in the sterile male described in this communication were found to be inherited when progeny were obtained by in vitro fertilisation and embryo transfer and, moreover, were transmissible through later generations by females. A genetic change is therefore clearly responsible for the phenotypic abnormalities. The mode of inheritance was clearly that of an autosomal dominant. The nature of the genetic change cannot be established with certainty but it seems likely that the 5;16 translocation identified cytologically was responsible. This is consistent with the reduced litter size of the 3 fertile females, with the low survival rate of the in vitro fertilised ova, and with the low sperm production of some males. The association of a translocation with a mutant phenotype is of interest as very few examples have been recorded in the literature (Searle, Personal communication). It may be significant that the homeobox mutant Hox-7, which is expressed not only in the nervous system but also in the neural crest, in the mandibular arch and derived mesenchymal tissues and at the apex of limb buds as they develop (Robert et al, 1988) lies in the vicinity of the chr 5 breakpoint and consequently, might be affected by the translocation and therefore responsible for the tremors and limb abnormalities seen in Ldn animals. REFERENCES 1. Fraser, L.R. & Drury, L.M. (1975) Biol. Reprod. 13:513-518. 2. Lyon, M.F. & Kirby, M.C. (1991) Mouse Genome 87:81-86. 3. Robert et a1 (1988) MNL 82 p 149. |
