A study, by means of Y-chromosome-specific repeated DNA probes, of mouse (ST) with small testes is reviewed. Mouse ST was shown to be a somatic mosaic of 10 % XY and 90 % XX cells. The cellular composition of the azoospermic testis reflected the overall proportions of XX and XY cells but it was found that XY cells predominated in the Sertoli cells of the testis tubules. These findings have been interpreted to indicate a fundamental role for the Sertoli cell in inducing testis organization in the indifferent gonadal rudiment, involving the expression of the Y chromosome

Jones, K. W.

Matsukuma, Shoichi

Singh, Lalji

English

Publications of the IAS Fellows

Development 101 Supplement. 143—149 (1987)
Printed in Great Britain © T h e Company of Biologists Limited 1987
143
The use of Y-chromosome-specific repeated DNA sequences in the
analysis of testis development in an XX/XY mouse
LALJI SINGH, SHOICH1 MATSUKUMA and K. W. JONES
Department of Genetics, University of Edinburgh, King's Buildings, West Mains Rd, Edinburgh EH9 3JN, UK
Summary
A study, by means of Y-chromosome-specific repeated
DNA probes, of mouse (ST) with small testes is
reviewed. Mouse ST was shown to be a somatic mosaic
of 10 % XY and 90 % XX cells. The cellular compo-
sition of the azoospermic testis reflected the overall
proportions of XX and XY cells but it was found that
XY cells predominated in the Sertoli cells of the testis
tubules. These findings have been interpreted to
indicate a fundamental role for the Sertoli cell in
inducing testis organization in the indifferent gonadal
rudiment, involving the expression of the Y chromo-
some.
Key words: Y-chromosome, mosaicism, mouse, testis
development, repeated DNA sequences, DNA. small
testes (ST).
Introduction
The role of the Y chromosome in testis development
remains to be clarified. However, we have obtained
some evidence (Singh, Matsukuma & Jones, 1987) to
suggest that the Y chromosome may be required in
the lineage that differentiates into the Sertoli cells.
Two DNA probes were used to detect the Y chromo-
some in the cells of an XX/XY mosaic mouse. One
probe (2(8)) consists of repeats of the tetranucleotide
GATA (Singh, Phillips & Jones, 1984), which has
previously been used to detect the sex-determining
pericentromeric region of the mouse Y chromosome
(Jones & Singh, \98\a.b; Singh & Jones, 1982).
However, since GATA sequences are also found
elsewhere in the mouse genome, a second mouse
Y-specific clone (M34), whose sequences are distrib-
uted only on the Y chromosome but outside the sex-
determining region, was also used. The study using
2[8] and M34 analysed the histological structure of
testis in a mosaic mouse (ST) containing only 10 % of
XY and 90% of XX cells.
The origin of the small testis mouse (ST)
The parents of ST developed from zygotes micro-
injected with Bkm-associated male-specific mouse
DNA in an independent study of experimental sex
reversal. ST, one of the second litter (4 males, 3
females), was found to have pale yellow small testes
(4x2mm) on dissection at the age of 30 days. Two
male litter mates dissected on the same occasion
showed normal-sized testes (6-0x4-0 mm,
7-0x4-0mm) and all other siblings (43 males, 52
females) were anatomically normal. Other anatom-
ical features of ST were normal, including accessory
sex organs and epididymis.
DNA analysis of the small testis mouse (ST)
Southern blots of ST liver DNA probed with 2(8)
showed hybridization in the high molecular weight
region (>23kb) similar to that of a normal male but
very much reduced in intensity (Fig. 1). This re-
duction could either have been due to inheritance of
integrated male-specific DNA from the microinjected
father in the absence of a Y chromosome, or to
mosaicism of XY and XX cells. To resolve these two
possibilities, ST DNA was probed with Y-specific
repeated sequence probe M34 (L. Singh & K. W.
Jones, unpublished data) which is not represented in
the sex-determining region of the mouse Y chromo-
some or in the male-specific DNA used for micro-
injection of the parents. The hybridization pattern of
M34 proved to be the same as that of control male
DNA, except that the intensity of the hybridization
was much reduced (not shown). This confirmed the
presence of a Y chromosome in the ST genome and
144 L. Singh, S. Matsukuina and K. W. Jones
suggested mosaicism as the cause of reduced intensity
of hybridization. The proportion of Y-bearing cells in
ST was estimated at 10% by comparison on a
Southern blot with DNA from normal female and
male mice mixed in various proportions (Fig. 2).
interphase nuclei but absent in control female prep-
arations. In contrast, out of a total of 140 chromo-
some preparations examined from ST, only 9 % of
euploid metaphases were found with high-density
grains in the pericentric region of the Y chromosome.
Chromosome analysis
To confirm the presence of a Y chromosome, bone
marrow chromosome preparations were examined
using;/; situ hybridization with 3H-labelled 2(8) probe
(Fig. 3). 100% (47/47) of control normal male prep-
arations showed the expected single concentrations of
autoradiographic grains in the pericentric region of
the Y chromosome, which was also visible in 83 % of
Histology of small testis
The testes from ST and a normal male litter mate
were fixed in formalin and processed for histological
study. The seminiferous tubules in the small testes on
both sides showed a single layer of cells within the
limiting membrane (Fig. 4A,B)- The cytoplasmic
processes of these cells extended into the lumen in
HaeUl _
A I 1 2 3 4
Alul.
6 7 8
kb
23-1-
9-4-
6-5- ** m
4-4-
23-
2-0-
056 -
ST ST
32 P-2(8)
Fig. I. Southern blot
hybridization of Alu\ and H<w\\\
restriction digests of 10/ig
aliquots of genomic DNA of ST
mouse and several male and
female litter mates with single-
stranded 32P-2(8) probe. Note
hybridization in the >23 kb
region of male DNA (tracks I
and 2) and its absence in female
DNAs (tracks 4. 5. 7 and 8). ST
DNA (tracks 3 and 6) shows
conspicuously reduced >23kb
hybridization.
the manner of Sertoli cells and, although one or
two degenerating cells which may have been sper-
matocytes were seen (arrow, Fig. 4B), sperm were
absent. In contrast, the normal control testes showed
well-developed seminiferous tubules filled with nu-
merous spermatogenic cells and contained some
sperm (Fig. 4C,D). The histiotypic composition of
the interstitial cell population appeared to be normal,
suggesting that Leydig cell differentiation was unaf-
fected. However, the interstitial cell population was
found to be considerably reduced compared with the
normal sib testis.
A sex mosaic mouse 145
The XY cell content of the small testis
To assess the contribution of XY cells to the testis of
ST, histological sections were studied by in situ
hybridization with clone M34. Hybridization due to
the Y chromosome was visible in about 70 % of cells
distributed at random in the normal control testis
sections (Fig. 5). This reflected the fact that 50% of
postmeiotic germ cells lack a Y chromosome and that
the condensation of the Y chromosome may vary in
different stages in chromosomal activity. In the ST
testis sections, a Y chromosome could be detected by
0 56-
32P-2(8)
Fig. 2. Experiment to determine the proportion of Y-bearing cells in the ST mouse by mixing various proportions of
male and female DNA for comparison with ST DNA after Alul digestion and 2(8) hybridization. Left panel shows the
ethidium-bromide-stained gel and HindlU size marker. Left panel: track 1, 10/jg ST DNA; track 2, 8^ /g female + 2//g
male DNA; track 3, 9 j.ig female + 1 ;ig male DNA; track 4, 9-5 jig female + 0-5;<g male DNA; track 5, 9-75 ^g
female + 0-25/Jg male DNA. Note the comparable intensity of >23 kb hybridization in tracks 1 and 3, indicating that ST
contains approximately 10% of Y-bearing cells.
146 L. Singh, S. Matsukuma and K. W. Jones
in situ hybridization in cells that were dispersed
amongst a majority population of Y-negative cells
(Fig. 6). consistent with the somatic proportion of
10% XY cells in the liver. However, the distribution
of cells in which the Y chromosome hybridized
unequivocally was found not to be random but to
predominate in the testis tubules in what appeared to
be Sertoli cells. Since in the normal sib no such
histological difference was observed in the distri-
bution of cells in which a Y chromosome could
definitively be detected, it was concluded that selec-
tion had occurred in favour of XY cells within tubules
of ST. It was impossible to determine whether this
represented preferential recruitment or survival of
XY cells. The proportions of XY cells in different
tubules appeared to be highly variable.
Discussion
Previous work has shown clearly that XX/XY chim-
aeras develop as males (McLaren. 1972). However,
much remains to be done in order to understand fully
Fig. 3. Chromosomes and mterphase nuclei of control normal male and ST male mouse hybridized in sun with
'H-labelled 2(S) probe. (A) A Y-bearing metaphase spread of ST mouse, note hybridization concentrated on the Y
chromosome. (B) Metaphase spread of normal male mouse, arrow points to Y chromosome. (C) Interphase nuclei of
ST mouse, note only one out of eight shows a cluster of grains compatible with the presence of the Y chromosome.
(D) Interphase nuclei of normal male mouse, note most nuclei show a cluster of grains indicating the Y chromosome.
A sex mosaic mouse 147
the effects of XX/XY mosaicism on testicular devel-
opment. As shown here, Y-chromosome-specific
probes can be useful in this type of study. The results
obtained with the ST mouse show that the somatic
proportion of XY cells, determined from the liver,
was reflected in the proportion of XY cells making up
the testis soma. This suggests that the uncommitted
embryo cell mass consisted of a random mixture of
XX and XY cells and that 10% of male cells is
sufficient to trigger testis development and to induce
basic testis architecture. This conclusion is consistent
with the rarity of hermaphrodite and female pheno-
types in experimentally produced XY-XX chimaeric
mice and supports a previous conclusion that most,
but not all, sex-chromosome chimaeras develop as
males (McLaren, 1972). Whether primordial germ
cells that migrate into the genital ridges at the time of
gonadal differentiation operate as the primary induc-
tor has not yet been settled in mammals. The histo-
logical structure of the testis tubules in the ST mouse,
however, suggested that the cellular content was
mainly of Sertoli cells together with a few poorly
defined germ cells in which it was not possible to be
certain of the genotype. There may have been inter-
ference with spermatogenesis by the environment
established by the predominating XX testis somatic
cells, consistent with previous findings of premature
meiosis and degeneration of XY germ cells in sex-
chromosome chimaeras (McLaren, 1972). Alterna-
tively, there may have been a scarcity of XY germ cell
precursors. XX cells are unable to undergo spermato-
genesis but oocytes have been reported in the testes
of sex-reversed XXv,r mice (McLaren, 1980). How-
ever, these do not persist and, since none were
Fig. 4. Histological sections of testes. (A) ST mouse showing single-layered seminiferous tubules. Note paucity of germ
cells and absence of spermatids and spermatozoa. (B) Higher magnification of A; arrows indicate degenerating cells
which may have been spermatocytes. (C) Normal litter mate of ST showing well-developed seminiferous tubules with
numerous spermatogonia. (D) Higher magnification of C.
148 L. Singh, S. Matsukwna and K. W. Jones
Fig. 5. Frozen section of testis of a normal male mouse
hybridized /// situ with 3H-labelled probe M34. Arrows
indicate clustering of grains in the nuclei of cells
containing a Y chromosome.
Fig. 6. Frozen testis section of ST mouse hybridized as in
Fig. 5 showing a minor population of XY (hybridization
to Y chromosome indicated by small arrows) cells
amongst XX cells. There is a predominant location of XY
cells within the testis tubules. The large arrow indicates
an area of interstitial cells lacking a Y chromosome.
positively identified in ST. it was suggested that they
may have already degenerated by the time this mouse
was examined.
Whilst there was no evidence of overall recruitment
of XY cells to form the testis itself, the proportion of
definitive XY cells was distinctly higher in testis
tubules when compared with the surrounding testis
interstitial cell population. This suggested either
preferential recruitment of Y-bearing cells during
tubule formation or their differential survival in the
tubule environment. This observation accords with
the fact that the sexual genotype of the cell is
functionally important for normal germline differen-
tiation, although it is not clear in which cells, or even
which tissue, the decisive influence of the Y chromo-
some is exercised. However, since the XY cells in the
ST testis sections were mainly Sertoli cells in the testis
tubules, these cells must be considered as the prob-
able locations in which the essential expression of
Y-chromosome primary sex genes occurs. This would
explain the longstanding observation that the forma-
tion of Sertoli cell cords appears to be the basic event
in testicular differentiation. Thus, whilst these obser-
vations on ST confirm that the Leydig cell can
differentiate in the absence of a Y chromosome, they
suggest that the presence of a Y chromosome may be
critically important for the differentiation and func-
tion of the Sertoli cell. The primary function of the
Y chromosome in directing the indifferent gonadal
primordium into the testis pathway may therefore be
expressed through the Sertoli cell precursors whose
differentiation may depend upon the presence of a
competent Y chromosome.
According to Eicher, Washburn, Whitney & Mor-
row (1982), the Y chromosomal gene Tdy suppresses
the pathway to ovarian differentiation and at the
same time activates testicular differentiation under
the control of autosomal sex genes. Since this regulat-
ory influence can extend to XX cells and be trans-
specific (Muller, Singh, Grund & Jones, 1982), the
Y-chromosomal regulatory gene must indirectly in-
fluence XX cells in the tissues of sex-chromosome
chimaeras and mosaics by an evolutionary con-
served pathway. Whether HY antigen is the paracrine
factor in testicular differentiation (Wachtel, Ohno,
Koo & Boyse, 1975) has been questioned by the
finding, in a variant of the sex-reverse mutation Sxr
(Sxr'), that HY antigen deficiency presents no barrier
to male development (McLaren et at. 1984). It is
therefore presently uncertain what induces XX cells
to participate in testis development.
The origin of the ST phenotype is unknown. Data
from liver DNA and in situ hybridization to testis
sections using the Y-specific probe M34 suggested
that all tissues may have consisted of the same
proportion of 90% XX and 10% XY cells. This may
A sex mosaic mouse 149
have come about either chimaerically by the fusion of
XX and XY embryos or by mosaicism. The former
explanation could invoke the fact that one or other
embryo cell population may be selectively discour-
aged (Tarkowski, 1961, 1963). The latter explanation
would require two events such as the founder cell of
the XX clone doubling its X chromosome while
simultaneously losing its Y chromosome.
The work reviewed here was carried out with the
financial assistance of MRC Grant No. G8306230CB to
K.W.J.
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The use of Y-chromosome-specific repeated DNA sequences in the analysis of testis development in an XX/XY mouse

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The use of Y-chromosome-specific repeated DNA sequences in the analysis of testis development in an XX/XY mouse

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