/. Embryol. exp. Morph. Vol. 36, 2, pp. 443-451, 1976
Printed in Great Britain
443
The effects of a t-allele (tAES) in the mouse
on the lymphoid system and reproduction
By M. VOJTISKOVA, 1 V. VIKLICKY, 1 B. VORACOVA,1
S. E. LEWIS 2 AND S. GLUECKSOHN-WAELSCH 2
From the Institute of Molecular Genetics, Czechoslovak Academy of Sciences
and Department of Genetics, Albert Einstein College of Medicine
SUMMARY
AEb
Mice homozygous for t , a recessive allele at the complex T-locus, are characterized
by their unique short-tailed phenotype as well as by runting and low fertility. Histological
and histochemical studies of the lymphoid and reproductive systems disclosed structural
changes in the mutant spleen resembling those found in autoimmune conditions. Involution
of the mutant thymus was greatly accelerated compared to normal. Necrotic changes occurred
during spermiogenesis whereas ovarian structure was normal in mutants. The possible mechanisms of the mutant effects are discussed in the framework of other similar syndromes and
the mode of action of alleles at the complex T-locus.
INTRODUCTION
A recessive allele at the complex T-locus, tAE5, with a unique homozygous
phenotype, was identified several years ago. tAE5 homozygotes are viable with
short tails, whereas homozygotes for any of the previously described recessive
f-alleles are either embryonic lethals or, if viable, have normal tails. In addition,
tAE5 homozygotes are runted with characteristically poor growth, viability and
fertility.
The syndrome of runting associated with low fertility, as well as preliminary
findings of morphological thymus deficiencies in tAE5 homozygotes, suggested
a possible autoimmune nature of the mutant condition. This possibility is
especially intriguing because of suspected involvement of the T-locus in the
control of cell surface properties (Gluecksohn-Waelsch & Erickson, 1970) and
the reports of serological identification of ^-associated cell surface components
with antigenic properties (Artzt & Bennett, 1975).
The studies reported here focus on the detection of histological and histochemical changes in the lymphoid system and gonads of tAE5 homozygotes. It
was hoped that such analysis might identify abnormalities in this particular
1
Author's address: Institute of Molecular Genetics, Czechoslovak Academy of Sciences,
Prague.
2
Author's address: Department of Genetics, Albert Einstein College of Medicine, New
York.
444
M. VOJTISKOVA AND OTHERS
mutant which would fit in with the proposed role of the T-locus in determining
cell surface antigens.
MATERIALS AND METHODS
AEh
The t
mutation arose in the mouse colony at Albert Einstein College of
Medicine in the phocomelic strain which itself was derived from a strain carrying the viable recessive allele t11 (Dunn & Gluecksohn-Waelsch, 1953). In the
course of breeding experiments with the phocomelic mutation, animals were
found with an exceptional short-tailed phenotype. These bred true for shorttailedness and were considered to be homozygous for a newly arisen recessive
/-allele, tAE5. For the histological analysis, 25 homozygous adult males (2-12
months old) and 17 females (newborn to 12 months old) were used. Mice from
a variety of inbred strains served as controls.
Organs to be examined were fixed in Carnoy's, embedded in paraffin, and
sectioned. Parallel sections of lymphoid organs, spleen and thymus, were stained
with haematoxylin-eosin, toluidine-blue (pH 2-0) or alcian blue. Parallel sections
of testes were stained with haematoxylin-eosin and Schiff reagent (Feulgen nuclear reaction), and sections of ovaries were stained with haematoxylin-eosin
only.
Activity of spermatogenesis was measured by determining the average frequency of tubules with maturing spermatozoa (Oakberg, 1956) in left and right
testes. After preliminary evaluation of sections from the central part of both
testes of two males in the first series, 13-15 pseudoserial sections from both
testes including about 2000 tubules were studied in the second series of five
males. The absence of appreciable variation in the state of spermatogenesis between centre and periphery indicated that the poor activity in mutant mice was
uniform and not of focal nature. Therefore, only 3-4 sections from peripheral
and central parts of each testis were evaluated in the remaining two series.
Sperm was stripped into normal saline from the vasa deferentia of four tAE5l
tAE5 m a i e s (3i_l0 months old) and three control males (2-7 months old).
Motility was scored qualitatively on a scale of 0 to + + + + . Viability was
assessed with the vital stain brilliant cresyl blue. Dry smears were prepared for
morphological study. The morphology of 200 sperm from each male was
studied with phase microscopy.
RESULTS
Breeding data
As mentioned earlier, tAEb homozygotes breed true for short-tailedness and
produce tailless (T/tAE5) and normal-tailed (+ jtAES) offspring when outcrossed
to Brachyury mice (77+). Breeding data are summarized in Table 1. The small
litter size in experiment 1 is a reflexion of the poor breeding performance of tAEb
homozygotes.
The effects of a t-allele in mouse
445
Table 1
Offspring
A
r
Expt.
Parents
1
tAE>jtAE5x
2
fAE5jfAE5 x Tj +
N
Short
tail
Normal
tail
No. of
mat ings
Total
no.
449
1557
0
1557
0
3-5
12
79
36
0
43
6-6
Tailless
Litter
size
Viability
tAEs homozygotes show drastically reduced viability at all ages. The small
litter size indicates possible prenatal death. In addition, in one experiment,
205 newborn out of a total of 331 were found to die within the first 48 h of life.
Survivors did not all reach weaning age. They appeared to be more susceptible to
slight infections than either wild-type mice or other mutants.
Weight
The average weight of homozygotes at weaning was 8 g compared to 15-20 g
for normal weanlings.
Histological results
Structural changes in the spleen
In mutant animals, the spleen could not be distinguished from normal (Fig. 1)
for the first two months of life; there was a clear distinction of the red and white
pulp with remnants of erythropoiesis and a gradual appearance of distinct
lymphoid follicles. In later stages the following abnormalities became noticeable in mutant mice: the incidence of megakaryocytes increased and signs of
activation became visible in the white pulp, e.g. germinal centres and distinct
perifollicular zones. Subsequently, a decline of activation as well as a decrease
of the white pulp area became apparent (Fig. 2). There seemed to be an increase
of proliferation of reticulum cells in the perifollicular zones, and later in the
entire spleen. The observed morphological changes resembled those resulting
from long-lasting antigenic stimulation which is first reflected in increased activation, later in proliferation of reticulum cells.
The proliferation of reticulum cells with increasing age results in the loss of the
normal spleen architecture, i.e. in the reduction of the white and replacement of
the red pulp, events known to be associated with a decline of functional capacity.
Structural changes in the thymus
The disintegration of cortical lymphocytes in the thymus of neonatal mutant
mice of both sexes presented a conspicuous difference from the thymus of
normal mice of several inbred strains. Nuclear debris and occasionally haematoxylin bodies were observed in newborn mutants (Fig. 3). At the age of 1-4
446
M. VOJTISKOVA AND OTHERS
Fig. 1. Normal spleen of 10-month-old mouse with distinct white pulp (W) and
residual erythropoeisis in the red pulp (E). Haematoxylin-eosin (HE), x 100.
Fig. 2. Spleen of a mutant 11-month-old female; follicles of the white pulp (W).
Note increased incidence of reticulum cells (R) in the perifollicular zones. HE, x 100.
Fig. 3. Thymus of mutant newborn female; numerous remnants of disintegrated
nuclei in the cortex, occasionally with formation of haematoxylin bodies (arrows). HE,
x700.
Fig. 4. Normal thymus of 2-month-old mouse with clear distinction of cortex (C)
and medulla (AQ. HE, x 220.
Fig. 5. Thymus of a 9-month-old mutant mouse with focus of mast cells (MC) in
parenchyma. Toluidine blue, at low pH. x 250.
Fig. 6. Pycnotic nuclei of mutant spermatogenic cells. Note swelling and vacuolization (arrows). HE, x 700.
The effects of a t-allele in mouse
447
months the development of the mutant organ was indistinguishable from normal
(Fig. 4), with a cortex thick and well distinguishable from the medulla in which
pseudo-Hassal's bodies were found in some cases.
In 3 to 4-month-old mutant mice occasional mast cells were observed in the
parenchyma; this cell type is absent at this age from the thymus parenchyma
of normal mice of various inbred strains (Viklicky, 1967). After the age of 6
months a change in the structure of the mutant organ was reflected in the reduction of the relative extent of the cortex and an increase in the incidence of mast
cells (Fig. 5) which are more numerous in mutants than in control animals of
the same age. The change of the cortex: medulla ratio in mutant mice is similar
to that observed in the normal thymus during physiological involution, but
in most cases it is much more extensive. In several such mice, the cortex was
virtually absent. This advanced involution of the thymus in mutant mice may
account for the fact that in many cases dissection failed to provide any thymic
tissue for histology but only hypertrophic parathymic lymphatic nodes.
Gonads
Reproductive performance is unusually poor in both male and female tAE5
homozygotes. This is expressed in failure of many such animals to reproduce at
all, in abnormally small litter size, high frequency of still-born young and premature termination of reproductive ability in breeding animals. For all these
reasons, it is very difficult to maintain the stock, and frequent outcrosses become
necessary.
Testicular histology and epididymal sperm
Table 2 summarizes the results of examination of testes of 25 homozygous
mutant adult males. Of the 25 males, the three oldest were included in the study
because they had proven to be fertile, as was the case for one of the 6-month-old
Table 2. Spermatogenesis and degree of degenerative changes of spermatogenic cells in t AE5 /t AE5 males
% of seminiferous tubules
with spermatozoa
A
No. of males
age (months)
Average
Range
2
10
5
5
3
2
3-3.5
4-4-5
5-6
9-12
14-3
15.0
16-2
13-6
9-2
13-4-16-6
3-4-23-0
10-5-21-9
9-2-18-8
8-1-14.3
Degree of
degenerative changes
+ to + +
+ to + +
+ to + +
++
- to +
males. Of the remaining males, one in the 3-month-old group was tested but
had failed to breed. The rest of the males were prepared for histological study
without having been tested for fertility. The table lists percentages of semini-
448
M. VOJTISKOVA AND OTHERS
ferous tubules containing spermatozoa, and degree of intensity of degenerative changes as none ( - ) , medium ( + ) and strong (-f +) in males of various
ages. Degree of spermatogenesis ranges from 3 % to 23 % in mutant males,
which is significantly below the range of 51-62 % reported for normal fertile
males of various inbred strains at the ages of 2-4 months (Pokorna, Vojtiskova,
Rychlikova & Chutna, 1963; Vojtiskova et al. 1965).
7' * i * * * *CT *
Fig. 7. Necrotic changes of mutant spermatogenic cells (arrows). Schiff staining.
(x 700).
Fig. 8. Section of mutant epididymis with spermatogenic cells of various stages,
giant multinucleated and necrotic cells (arrows). HE, x 450.
Various degenerative phenomena were conspicuous in almost all mutant
males. First of all, the incidence of binucleate and giant multinucleate cells
seemed to exceed that observed in normal spermatogenesis, indicating a possible increase of defects of cell division in the mutant. An additional and
even more frequent difference from normal was the observation of necrotic
changes in mutants. The cells had become enlarged and pale. Cell outlines had
disappeared and there was much evidence of cellular fusions. Some nuclei
appeared shrunken and pycnotic while others were swollen and vacuolated and
had lost their capacity to be stained with basic dyes (Figs. 6, 7). The necrotic
changes seemed to concern primarily the spermatids, in various stages of spermiohistogenesis. They were most severe in tubules with the maximum frequency
of spermatids and maturing spermatozoa; besides apparently normal spermatids
there were many necrotic cells with nuclei of bizarre shape, sometimes resembling huge spermatid nuclei (Fig. 6). Not infrequently necrotic late spermatids
still attached to the Sertoli cells seemed to fuse with each other.
Finally, sections of the mutant epididymi showed the passing into the
ductus of precursor cell stages (Fig. 8), as well as the normally expected mature
spermatozoa.
The effects of a t-allele in mouse
449
AE5 AE5
Viability of t /t
sperm did not differ from that of control sperm. Most
of the tAE5/tAE5 sperm had normal morphology although the sperm strips from
tAE5jfAE5 m a i e s contained more spermatocytes and amorphous cellular debris
than did those from control males. The increased frequency of cellular debris in
tAE5jtAE5 S p e r m strips correlates well with the necrotic changes in spermatogenic
cells observed histologically. Motility of sperm from tAE5/tAE5 males ranged from
+ to + + + while that of normal males was uniformly + + + + .
Ovaries
In none of the 14 females examined did the ovaries display any gross morphological deviations. Ovaries of adult females (4-12 months old) included all
developmental stages of Graafian follicles and corpora lutea. Thus, in contrast
to males, the poor reproductive performance of mutant females was not reflected in the morphology of the gonads.
DISCUSSION
Abnormalities of the lymphoid system and of spermatogenesis were shown
to characterize mice homozygous for the recessive allele tAE5 at the T-locus,
which have low viability and fertility. Thymus and spleen are severely affected
in both sexes, in contrast to the gonads which are abnormal in the male only.
The interpretation of the abnormalities reported here must take into account
the fact that the majority of tAm homozygotes do not survive past weaning
age. Therefore the adult animals studied represent a group selected because of
their survival. It is probable that this genotype is in fact responsible for lethal
defects more severe than those described here.
The association of runting, low viability, deficiencies of the lymphoid system and of reproduction in tAEh homozygotes is not unique but may be found
in certain other genetic defects in mice. For example, reduction of growth rate,
low fertility and an underdeveloped reproductive system are characteristic of
'nude' mice with thymus dysgenesis and a deficient immune response (Flanagan, 1966; Pantelouris & Hair, 1970; Wortis, Nehlsen & Owen, 1971; Pritchard &
Micklem, 1972; Pritchard, Riddaway & Micklem, 1973; Shire & Pantelouris,
1974). Furthermore, the genetically complex NZB mouse shows structural
changes in spleen and thymus in both sexes, in addition to reproductive
deficiency in males (Bielschowsky, Helyer & Howie, 1959; Yiklicky & Polackova,
1969; Fernandes, Yunis & Good, 1973). Finally, experimentally induced autoimmune aspermatogenesis is associated with morphological abnormalities of the
lymphoid system (Vojtiskova et al. 1965). Although^ 5 homozygotes were not
tested for their immune capacity, their viability might be suggestive of a
deficient immune responsiveness.
Whereas mutant ovaries appeared normal, spermatogenic activity was
found to be reduced severely. The minor defects observed in mutants during
450
M. VOJTISKOVA AND OTHERS
meiosis do not seem to block spermatogenesis which is able to proceed to the
spermatid stage. However, though present in at least normal numbers, spermatids frequently failed to differentiate into functional spermatozoa in the mutant
homozygote; they degenerated and died at various stages of spermiohistogenesis.
In this respect, the effects of the tAEb allele on male reproduction appear to differ
from those of previously studied ?-alleles (Braden, 1973; Gluecksohn-Waelsch,
1972; Braden et al 1972), but might resemble those of tw2 (Dooher & Bennett,
1974). It is interesting that yet another ^-allele has now been shown to have an
effect on male reproduction.
The possible relationship of the pathological changes in the lymphoid system
to the deficient reproductive function is unknown. There are some indications
that in the nude mouse the correction of the defective immune capacity (Wortis,
Nehlsen & Owen, 1971) also tended to improve reproductive function (Shire &
Pantelouris, 1974).
The complex of developmental defects in tAEb homozygotes finds a parallel
in experiments where administration of a steroid anti-androgen caused abnormalities in both spermatogenesis and the lymphoid system (Polackova, Viklicky & Vojtiskova, 1975). The resulting phenocopies point to androgenregulated developmental processes as possible targets of both the anti-androgen and the mutated /-allele. Thus, it is possible that a hormonal defect might
be implicated in the genetic abnormality.
The effects of tAE5 on thymus, spleen and testes are compatible with a previously expressed interpretation of T-locus effects on development and differentiation. It was suggested (Gluecksohn-Waelsch & Erickson, 1970) that the
T-locus may control cell surface properties involved in cell-to-cell recognition,
cell interactions and morphogenetic movements during embryogenesis. In tAEb
mutants it cannot yet be decided whether such cell-surface properties involve the
action of hormones, or other aspects of cell recognition and interactions.
The authors thank Howard A. Turchin for excellent assistance in the preparation of the
illustrations. This study was supported by grants from the National Institutes of Health
HD-00193, GM-00110, GM-19100, and by a grant from the American Cancer Society,
VC-64.
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{Received 6 May 1976, revised 2 July 1976)