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X-chromosome monosomy in the myelin-deficient rat mutant.

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THE ANATOMICAL RECORD 226:396-402 (1990)
X-Chromosome Monosomy in the Myetin-Deficient
Rat Mutant
Departments of Physiology and Rehabilitation Medicine, and Cytogenetics Laboratory,
Department of Pathology, N.Y.U. School of Medicine, New York, New York
We have identified three examples of female Wistar rats exhibiting the tremor and seizures characteristic of the X-linked myelin deficiency (md)
mutation, which is ordinarily seen only in males. Cytogenetic study of two of these
animals has shown them to have 41 chromosomes instead of the normal 42. The
missing chromosome was identified a s a n X chromosome by G-banding analysis.
These animals thus have a n XO genotype comparable to that in Turner’s syndrome. Anatomically, one of the animals, which was studied in detail, showed no
abnormality of the uterus, and the ovaries, although somewhat smaller than normal, were histologically indistinguishable from those in a normal female rat. No
evidence of endocardial fibroelastosis was detected, nor was there any anomaly of
the aorta. The myelin deficiency in the central nervous system was comparable to
that in hemizygous mutant male rats. XO monosomy in the Wistar rat thus has
little effect on phenotype and is more comparable to that in mice than to Turner’s
syndrome in man. The myelin-deficient rat is useful for studies of X-chromosome
monosomy since XO females can readily be identified by the neurological syndrome characteristic of the md mutation.
The myelin-deficiency (md) mutation is a n X-linked
recessive and thus appears in one-half the male offspring of heterozygous female carriers. Affected males
are easily identified by the characteristic action tremor
that develops a t 2 weeks of age, followed by tonic seizures a t 3 weeks and death by 4 weeks.
In the course of breeding these animals, we have
found three examples of females that also displayed the
tremor and seizures typical of this mutation. One died
before it could be studied, but the other two have been
analyzed cytogenetically, and one of them has been
studied by gross anatomical and histological methods.
The results of these studies show that the female md
rats (mdr) are examples of X-chromosome monosomy,
comparable to Turner’s syndrome in humans, and that
the neurological and neuropathological defects in these
animals are comparable to those in hemizygous male
littermates (Dentinger et al., 1982; Rosenbluth, 1985,
Two mdr females, the offspring of Wistar female carriers bred with normal Wistar males, were anesthetized with 0.1-0.2 ml 10% chloral hydrate, and samples
were taken under sterile conditions from multiple organs, including the liver, lung, kidney, spleen, omentum, diaphragm, skeletal muscle, and skin. The tissues
were finely minced, suspended in Dulbecco’s modified
Eagle medium (supplemented with 20% fetal calf serum, 1% penicillin-streptomycin, and 1% glutamine),
plated into tissue culture flasks, and incubated at 37°C
in a 5% CO2 atmosphere. The cultures were observed
daily for evidence of mitotic growth and were harvested
for chromosome analysis within 1-2 weeks. Cells were
exposed to colcemid (final concentration 0.1 mg/ml) for
3 hr, treated with 0.075 M KC1 for 20 min at 37”C, and
fixed in three changes of methanol-acetic acid (3:l).
Slides were prepared by air drying and were aged a t
60°C for 24-28 h r before staining with Giemsa-trypsin,
as described previously (Benn and Perle, 1986). Gbanded metaphase spreads were analyzed microscopically and photographed. Thirty metaphase spreads
were counted, and five cells were karyotyped from tissues from each animal. Chromosome classification was
based on the numbering system determined by the
Committee for a Standardized Karyotype of Rattus
noruegzcus (1973).
In one 20-day-old animal, immediately following removal of the tissue samples for cytogenetic analysis,
fixation was carried out by transcardiac perfusion with
3%glutaraldehyde and 2% formaldehyde in 0.1 M cacodylate buffer (pH 7.41, supplemented by injection of
fixative directly into the brain. The spinal cord, ovaries, heart, and aorta were dissected, examined grossly,
and photographed; samples of these tissues were then
postfixed with 1%osmium tetroxide in 1.5% potassium
ferrocyanide, dehydrated, embedded in Araldite, thinsectioned, and stained for electron microscopy by standard methods. For routine histological analysis 1 pm
sections were stained with 0.5% toluidine blue in 1%
sodium borate. One micrometer sections were stained
with a modification of the method of Musto (1981) for
Received March 9, 1989; accepted J u n e 5, 1989.
Address reprint requests to Dr. J. Rosenbluth, RR 714, N.Y.U.
School of Medicine, New York, NY 10016.
the ventricles (Fig. 5). Thus we found no evidence of
endocardial fibroelastosis in the female mdr.
Examination of the spinal cord demonstated typical
mdr morphology. Spinal roots were normally myelinated (Figs. 6, 81, but the spinal cord itself was virtually devoid of myelin, in obvious contrast to normal
controls (Figs. 7, 9). Cross-sections through the spinal
cord showed the lateral and ventral fiber tracts and the
dorsal columns to be composed almost entirely of unmyelinated axons (Figs. 6, 8). Occasionaly myelinated
segments were encountered (Figs. 8, 101, but, a s with
male mdr (Rosenbluth, 19871, thin sections displayed a
variety of abnormalities and irregularities in these
sheaths as well as dilatation of cisternae of the granular endoplasmic reticulum in oligodendrocytes (Fig.
Fig. 1. Photograph of female mdr pelvic viscera. The bicornuate
uterus is indicated by arrows.
demonstration of elastic fibers. In brief, sections dried
onto glass slides were immersed directly in the staining solution (2%Weigert’s hematoxylin and 2% iodine1
4%potassium iodide), rinsed, and immersed in 5%PTA
and then 1% acetic acid, and finally air dried and
mounted. No counterstain was used. Two control animals, one male mdr and one normal female, were anesthetized with chloral hydrate, a s described above, and
perfused with 4% formaldehyde in cacodylate buffer
(pH 7.4). After the viscera were photographed, the spinal cord, heart, and ovaries were postfixed, embedded,
sectioned, and stained for comparison with the counterpart tissues taken from the female mdr.
A total of 30 G-banded metaphase cells was analyzed from each of the two female mdr. Cells from two
different tissue types were examined to rule out sex
chromosome mosaicism. The total chromosome number
in all cells was 41 (Fig. ll),in contrast to the normal
diploid number of 42 chromosomes seen in metaphase
cells analyzed from a phenotypically normal female littermate.
In both animals, intrachromosomal (GTG)-banding
patterns revealed the sole abnormality to be the absence of one sex chromosome; i.e., all the cells examined contained only a single X chromosome (Fig. 11).
No structural aberrations were noted, no cells were
found with two X chromosomes, and no Y chromosomes
were found in any of the cells. Thus no evidence of
chromosome mosaicism or translocation was detected
in these studies. The results indicate sex chromosome
monosomy in these animals similar to that seen in
Turner’s syndrome in human females.
Animals were identified a s female by the characteristic short anogenital distance. Examination of the viscera in the one female mdr studied in detail showed a
typical bicornuate uterus (Fig. 1). In contrast to the
ovarian agenesis characteristic of Turner’s syndrome
in humans (Wilkins and Fleischmann, 19441, both ovaries were clearly present, although they appeared
smaller than normal. Histologic study (Fig. 2) showed
normal-looking germ cells and follicles. Numerous epitheloid cells in the surrounding stroma presumably
represent the interstitial gland, involved in steroid secretion. Microscopically, the ovary of the female md rat
was equivalent to that in the normal female control r a t
(Fig. 3).
In view of reports of cardiac and aortic abnormalities
in Turner’s syndrome in humans (see Robbins et al.,
1984, for summary), the heart and aorta of the female
mdr were examined but showed no signs of coarctation,
stenosis, or other gross abnormality. On histological
examination, no thickening of the endocardium was
seen in comparison with controls. Sections stained for
elastic fibers showed a clear-cut internal elastic lamina
in arterioles (Fig. 4) and some elastic fibers in the endocardium of the atria, but none in the endocardium of
Although sex chromosome monosomy is known to
occur in a variety of mammals (see Wurster-Hill et al.,
1983, for review), there may be no obvious external
characteristics to identify such animals, and presumably it is for this reason that there have been few
previous reports of such occurrences in rats. The md
animals offer a n advantage in identifying this abnormality in that the neurological defect is unmistakable
and can be used as a marker. Since the md mutation is
clearly X-linked, finding a female mutant immediately
suggests numerical or structural aberrations of the X
chromosome, a supposition that we have now confirmed in two cases in which cytogenetic analysis has
shown 41 chromosomes, with only a single X chromosome, instead of the normal 42.
XO females have been identified previously among
other rats. With the Asian type (2n = 42 chromosomes), Yong (1971) found three examples of 41 chromosome, XO females, and Sharma and Raman (1971)
also identified a n XO female. Chromosome analysis in
both reports was on unbanded cells, however; thus the
X chromosome loss was not confirmed by banding analysis. In the Oceanic-type black rat (2n = 38 chromosomes), examples of XO females have been described by
Yosida et al. (1974), Satya Prakesh and Aswathanarayana (1977), and Yosida (1977, 1979). The studies
Fig. 2. Photomicrograph of ovary from female mdr. A follicle containing an ovum (Ov) is shown. Interstitial gland cells (I) containing
fine granules surround the follicle. G, granulosa cells. x 580.
Fig. 3. Photomicrograph of a follicle in a normal rat ovary showing
an ovum, granulosa cells and interstitial cells labeled as in Figure 2.
x 580.
Fig. 4. Photomicrograph of an arteriole stained for elastic fibers in
cardiac ventricle of a female mdr. The internal elastic lamina is conspicuous (arrow). x 650.
Fig. 6. Photomicrograph of female mdr spinal cord. The ventral root
(arrow) contains myelinated fibers, but the cortical fiber tracts within
the cord are virtually devoid of myelin. x 120.
Fig. 5. Cardiac ventricle of female mdr stained for elastic fibers. The
endocardial surface (arrow) shows no staining. x 880.
Fig. 7. Photomicrograph of normal rat spinal cord showing myelination of ventral root (arrow) and cortical fiber tracts. Y 120.
Fig, 11. Representative G-band karyotype of a metaphase cell from female mdr (41,X). Partial karyotypes are provided (above those from the original cell) of chromosome pairs 3, 13, and 18 to resolve
unclearly banded regions.
Fig. 8. Detail of Figure 6 (female mdr spinal cord) showing normally
myelinated ventral root (top) and unsheathed axons within spinal
cord fiber tracts. Occasional abnormal myelin sheaths are present
(arrows). x 1,200.
Fig. 9. Detail of Figure 7 (normal spinal cord) showing myelination
of ventral root (top) and cortical fiber tracts. x 1,200.
Fig. 10. Electron micrograph of female mdr spinal cord fiber tract
showing a n abnormal oligodendrocyte (OL) containing markedly dilated cisternae of granular endoplasmic reticulum. Several axons (*I
are surrounded by abnormal myelin sheaths. x 24,000.
by Yosida and coworkers used G-banding analysis to
characterize fully the presence of X-chromosome monosomy.
Most of the published studies on XO rats indicate no
visible external abnormalities, but little information is
available on organ pathology. Yong (1971) reported no
abnormalities of the reproductive organs on autopsy,
and Sharma and Raman (1971) reported smaller than
normal ovaries. However, no histological studies were
done in either case. In the one example we studied in
detail, there was no sign of endocardial fibroelastosis or
the other cardiac or aortic abnormalities sometimes associated with Turner’s syndrome in humans, and the
ovaries were histologically normal. Thus XO rats are
similar to XO mice, which not only have normal ovarian tissue but are also fertile (see Green, 1981, for review).
With regard to the myelin deficiency, the spinal cord
in the female mdr examined appeared to be identical to
that of male mdr studied previously (Rosenbluth,
1987). The absence of a Y chromosome had no apparent
effect on the severity or expression of the pathological
lesion or on the neurological consequences. The female
mdr, like their male counterparts, developed the typical action tremor and then generalized tonic seizures at
the expected times. Presumably, in them, as in the
males, the absence of myelin leads to spontaneous activity of central axons either as a result of ephaptic
transmission of impulses among neighboring axons at
sites of sodium channel accumulation or because of excess extracellular potassium accumulation resulting
from continuous, a s opposed to saltatory, conduction in
the unsheathed axons (see Rosenbluth, 1988, for discussion). Such activity originating in central nervous
system fiber tracts presumably leads to the seizures
seen (Rosenbluth, 1985) except when this activity is
experimentally prevented from spreading to higher
centers (Rosenbluth and Hasegawa, 1988).
In summary, using the characteristic neurological
syndrome, consisting of a n action tremor developing at
2 weeks and tonic seizures developing at 3 weeks, as a
marker, we have identified two XO females among md
Wistar rats. These animals are more similar to XO
mice than to humans with Turner’s syndrome in that
normal ovarian tissue is present, and there are no abnormalities of other organs. The neurological and neuropathological defects are identical to those in male
mdr .
This study was supported by grants from the NIH
(NS 07495) and the Multiple Sclerosis Society (RG1579). The authors are indebted to Ron Morella for
expert technical assistance.
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monosomy, deficiency, rat, myelin, mutant, chromosome
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