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Introduction
The first critical steps in testicular development from the
indifferent gonad and subsequent masculinization of the
fetus are differentiation of the Sertoli cells and Leydig cells
(Byskov, 1986; Magre and Jost, 1991). Resultant formation
of the male reproductive tract and degeneration of the
female tract depends on normal development and function
of these cell populations (Jost
et al.
, 1973). Currently, we
lack a full understanding of the factors that normally
regulate the numbers of Sertoli and Leydig cells during fetal
and neonatal life, although it is clear that this is a critical
period for subsequent development of normal reproductive
function (Orth
et al.
, 1988; Lejeune
et al.
, 1998).
Gonadotrophins regulate the activity of pubertal and
post-pubertal populations of Sertoli cells and Leydig cells
(Griswold, 1993; Lejeune
et al.
, 1998), although the role of
these hormones in determining the number of testicular
cells during the fetal and neonatal periods remains unclear.
Evidence from earlier studies in rats and sheep indicates that
proliferation of Sertoli cells is at least partially
gonadotrophin-dependent during fetal life (Orth, 1984;
Thomas
et al.
, 1994), although the stage at which
dependency begins is not known. In contrast, there have
been no studies to determine whether Leydig cell
proliferation and differentiation are gonadotrophin-
dependent during the fetal and neonatal period, although
O’Shaughnessy
et al.
(1998) showed that Leydig cell
function is independent of pituitary control during this time.
The aim of the present study was to measure Leydig cell,
Sertoli cell and gonocyte numbers during fetal and postnatal
development in normal mice and in hypogonadal (
hpg
)
mice, which lack GnRH, and, therefore, endogenous
Role of gonadotrophins in regulating numbers of Leydig and
Sertoli cells during fetal and postnatal development in mice
P. J. Baker and P. J. O’Shaughnessy*
Division of Veterinary Physiology and Pharmacology, Department of Veterinary Preclinical
Studies, University of Glasgow Veterinary School, Bearsden Rd, Glasgow G61 1QH, UK
Reproduction
(2001) 122, 227–234
Research
The role of the gonadotrophins in regulating numbers of
Leydig and Sertoli cells during fetal and postnatal develop-
ment was examined using normal mice and hypogonadal
(
hpg
) mice, which lack circulating gonadotrophins. The
disector method was used to determine the number of cells
from day 16 of gestation until adulthood. The numbers of
Leydig cells did not change significantly between day 16 of
gestation and day 5 after parturition in normal mice and
were not significantly different from numbers in
hpg
mice
at any age up to day 5 after parturition. There was a 16-fold
increase in the number of Leydig cells in normal mice
between day 5 and day 20 after parturition, followed by a
further doubling of number of cells between day 20 and
adulthood. The number of Leydig cells in
hpg
testes did not
change between day 5 and day 20 after parturition but
doubled between day 20 and adulthood so that the number
of cells was about 10% of normal values from day 20
onwards. Leydig cell volume was constant in normal
animals from birth up to day 20 and then showed a 2.5-fold
increase in adult animals. Leydig cell volume was normal
in
hpg
testes at birth but decreased thereafter and was
about 20% of normal volume in adult mice. The number of
Sertoli cells increased continuously from day 16 of
gestation to day 20 after gestation in normal mice and then
remained static until adulthood. The number of Sertoli
cells in
hpg
testes was normal throughout fetal life but was
reduced by about 30% on day 1 (day of parturition).
Thereafter, Sertoli cells proliferated at a slower rate but
over a longer period in the
hpg
testis so that on day 20 after
parturition the number of Sertoli cells was about 50% of
normal values, whereas in adult mice the number was 65%
of normal. The number of gonocytes did not change
between day 16 of gestation and day 1 and did not differ
between normal and
hpg
testes. The number of gonocytes
increased nine-fold in normal testes but only three-fold in
hpg
testes between day 1 and day 5 after parturition.
Gonocytes differentiated into spermatogonia in both
normal and
hpg
testes between day 5 and day 20 after
parturition. These results show: (i) that fetal development
of both Sertoli and Leydig cells is independent of
gonadotrophins; (ii) that normal differentiation and
proliferation of the adult Leydig cell population (starting
about day 10 after parturition) is dependent on the
presence of gonadotrophins; and (iii) that the number of
Sertoli cells after birth is regulated by gonadotrophins,
although proliferation will continue, at a lower rate and for
longer, in the absence of gonadotrophins.
© 2001 Journals of Reproduction and Fertility
1470-1626/2001
*Correspondence
Email: P.J.OShaughnessy@vet.gla.ac.uk
circulating gonadotrophins (Cattanach
et al.
, 1977). These
data demonstrate the stages of development at which
proliferation of each cell type becomes gonadotrophin-
dependent and the role of the gonadotrophins in determin-
ing the number of cells at puberty.
Materials and Methods
Animals
Normal and
hpg
mice were bred at the University of
Glasgow Veterinary School from stock derived originally
from the Oxford breeding colony. Animals were maintained
as required under United Kingdom Home Office regulations
as applied to the use of experimental animals. For timing of
fetal development, males were caged overnight with
females and the morning was designated as day 0.5 of
gestation. For studies on post-natal animals the day of birth
was designated as day 1. Adult animals were aged between
70 days and 120 days. Normal and
hpg
mice were
distinguished before puberty by PCR as described by Lang
(1995).
Fixation and processing
For cell counting using the physical disector method (see
below) testes were fixed by immersion in 2.5% (w/v)
glutaraldehyde, 2% (w/v) paraformaldehyde and 0.1% (w/v)
picric acid in 0.1 mol cacodylate buffer l
–1
(pH 7.4). After
osmication and dehydration, each testis was embedded in
Technovit 7100 (Kulzer and Co, GmbH, Wehrheim) and cut
into sections (2 µm thickness). Every tenth or twentieth pair
of sections was mounted and stained with toluidine blue in
1% (w/v) borax. For cell counting using an optical disector
(see below) testes were fixed in Bouin’s fluid overnight,
placed in 70% (v/v) alcohol and embedded in Technovit
7100 resin. The testes were cut into sections (20 µm
thickness) and stained with Harris’ haematoxylin.
Sterology
Two methods of cell counting were used. Most testes
were analysed using the physical disector method, but
towards the end of the study equipment and software were
obtained to allow the optical disector to be used. The two
methods provide comparable data (Wreford, 1995 and the
present study) but the optical disector is considerably faster.
Total testis volume was estimated for both methods using
the Cavalieri principle (Mayhew, 1992) and the slides used
to estimate the number of cells were also used to estimate
testis volume to avoid any requirement for correction
factors due to tissue shrinkage. A computer running Auto-
CADlt97 software (Autodesk Inc, San Rafael, CA) and a
digitizing tablet were used to estimate the surface area of
selected sections (every tenth or twentieth section). The
total surface area of these selected sections is designated
SA
. The thickness of each section is known (
h
), as is the
distance between the sections (
d
), and so total testis volume
(
T
v
) can be calculated by
T
v
=
SA
⫻
h
⫻
d
. For the physical
disector the method used was based on previous
descriptions by Sterio (1984) and Gundersen (1986). A Leitz
Laborlux S microscope with an attached drawing tube was
used to view randomly selected areas of testis with a ⫻ 100
oil immersion objective. The drawing tube allowed the
outline of the nuclei present to be drawn onto a disector
counting frame enclosing an area of 4500 µm
2
. The same
area was located in the adjacent section and the nuclei
were drawn onto an acetate sheet. Nuclei present in one
image but not in both were then counted. A running mean
of the number of nuclei present in each pair of drawings was
calculated and cell counting was continued until the
standard deviation coefficient of the means of the last 50
measurements was < 5%. The optical disector technique
(Wreford, 1995) was used to count the number of Leydig
and Sertoli cells in the testes of normal and
hpg
adult mice.
The numerical density of each cell type was estimated using
an Olympus BX50 microscope fitted with a motorized stage
(Prior Scientific Instruments, Cambridge) and Stereologer
software (Systems Planning Analysis, Alexandria, VA).
The volume density of Leydig cells was determined by
the point-counting method using the Stereologer program.
Testis sections were cut (2 µm thickness) and the software
firstly selected the areas of tissue to be counted and then
superimposed a 121 point grid over a video image taken
with a ⫻ 100 objective lens. At least 100 grids were counted
for each testis measured. The mean Leydig cell volume was
determined by multiplying the volume density by the total
testis volume and dividing by the total number of Leydig
cells.
In all studies, gonocytes, Sertoli cells and Leydig cells
were identified as described by Hardy
et al.
(1989),
Vergouwen
et al.
(1991) and Duckett
et al.
(1997).
Statistical analysis
Results were analysed by two-way ANOVA followed by
comparison of individual means using
t
tests.
Results
Testis volume
Changes in testis volume during development in normal
and
hpg
mice are shown (Fig. 1). There was no difference in
testis size between the two groups until birth. After birth,
testis volume in normal mice continued to increase rapidly,
in contrast to the
hpg
mice, which showed a much slower
growth rate. By adulthood the volume of the
hpg
testis was
about 1.5% of that of normal mice.
Leydig cells
Fetal Leydig cells were identified by their rounded nuclei
and distinct, darkly stained cytoplasm, which sometimes
contained lipid droplets (Fig. 2a–d). Up to day 1 (day of
parturition) the cells appeared singly or in small groups and
228
P. J. Baker and P. J. O’Shaughnessy
there were no clear differences in morphology between
normal and
hpg
mice. On day 5 after birth, larger clusters of
Leydig cells began to appear in normal animals and on day
20 clusters of cells with a more variable nuclear morphol-
ogy and less distinct cytoplasmic boundaries were observed
(Fig. 2e,g). These cells frequently contained small lipid
droplets (Fig. 2g) and had a similar morphology to cells in
adult mice (not shown). In
hpg
testes the Leydig cells after
day 5 were characterized by the presence of large lipid
droplets as described by O’Shaughnessy and Sheffield
(1990) (Fig. 2h,i). Leydig cells usually appeared singly in
hpg
testes after day 5 (Fig. 2h) except near the rete testis
where clusters of Leydig cells were observed (Fig. 2i).
Leydig cell volume did not change in normal mice
between birth and day 20 but then increased approximately
2.5-fold to adulthood (Fig. 3). Leydig cell volume in
hpg
mice was normal at birth but decreased to about 50% of
normal values by day 20 after parturition. There was no
hypertrophy of Leydig cells in adult
hpg
mice and the
volume of these cells was about 20% of normal volume in
adult mice.
There was little or no change in the number of Leydig
cells during late fetal or early neonatal life in normal mice
(Fig. 4). However, between day 5 and day 20 after birth, the
number of cells increased 16-fold followed by a further
doubling between day 20 and adulthood (Fig. 2). In
hpg
mice, the number of Leydig cells was not significantly
different from that in normal mice throughout fetal life and
up to day 5 after birth. After day 5, the number of Leydig
cells increased slightly in
hpg
mice up to day 20 and then
doubled between day 20 and adulthood so that on day 20
and in adult mice the number of Leydig cells in
hpg
mice was about 10% of normal values.
Sertoli cells
During fetal development the Sertoli cells were clearly
identifiable within the developing tubules of normal and
hpg
mice. The nuclei were of variable sizes and shapes and
were located towards the periphery of the tubules (Fig.
2a–d). In early neonatal life, the Sertoli cell nuclei tended to
become more central and by day 20 after birth they had
assumed an adult morphology in normal mice (Fig. 1e).
There were no clear morphological differences between
Sertoli cells from normal and
hpg
mice.
The number of Sertoli cells in normal mice increased
rapidly throughout late fetal life and early post-natal life
with a 6.5-fold increase in number between day 16 of
gestation and birth and a four-fold increase between birth
and day 5 (Fig. 4). After day 5, the rate of proliferation
decreased and there was a 1.9-fold increase in the number
of Sertoli cells between day 5 and day 20, with no further
increase thereafter. The number of Sertoli cells was normal
in
hpg
mice during fetal life up to day 18 of gestation but
significantly lower at birth (Fig. 4). After birth, the number of
Sertoli cells continued to increase in the
hpg
mice but at a
slower rate than in normal mice, so that by day 5 and day 20
after birth the numbers of Sertoli cells in
hpg
mice were
51% and 48% of normal values, respectively (Fig. 4).
However, proliferation of Sertoli cells in the
hpg
testes
continued beyond day 20 after birth, so that by adulthood
the number of Sertoli cells was about 65% of normal values
(Fig. 4).
Gonocytes
The gonocytes in normal and
hpg
testes were clearly
identifiable in the fetal testes with large round nuclei, often
surrounded by a distinct cytoplasm, and situated within the
lumen of the tubules. The gonocytes were clearly distin-
guishable from the nuclei of the Sertoli cells (Fig. 2). Some
cells had migrated to the basement membrane of the
tubules by day 5 after birth and by day 20 the gonocytes had
differentiated into spermatogonia in both normal and
hpg
testes (Fig. 2e,f). Further differentiation and development
along the spermatogenic pathway was significantly greater
at day 20 in normal mice compared with
hpg
mice (Fig.
2e,f ).
Gonocyte numbers did not change between day 16 of
gestation and birth in normal mice (Fig. 4). There was a
nine-fold increase in the number of gonocytes in normal
testes between day 1 and day 5. In the
hpg
mice, gonocyte
numbers were not significantly different from normal values
during fetal life. There was an increase in gonocyte numbers
in
hpg
mice between day 1 and day 5 after birth, but the rate
of proliferation was lower than in normal mice and gono-
cyte numbers were reduced significantly on day 5.
Discussion
Normal masculinization of the fetus and fertility in adult life
depend on differentiation and development of Leydig cells
and Sertoli cells during fetal and neonatal life. The results of
the present study show that, during the fetal period,
differentiation and proliferation of both Sertoli cells and
Leydig cells are largely independent of endogenous gonado-
trophins. After birth, normal development of the number of
cells becomes gonadotrophin-dependent in both cell types
Regulation of numbers of Leydig and Sertoli cells
229
0
1
2
3
20
40
60
80
100
120
16 18 1 5 20 Adult
Day of
gestation
Day after birth
Testis volume (mm
3
)
Fig. 1. Testis volume during development in normal (䊏) and
hypogonadal (
hpg
) (䊐) mice. Values are mean ⫾ SEM.
230
P. J. Baker and P. J. O’Shaughnessy
(a) (b)
(c) (d)
(e) (f)
(g) (h)
(i)
Fig. 2. Photomicrogaphs of testicular tissue from mice of different ages stained with
toluidine blue. (a) Normal mouse on day 1 (day of birth); (b) hypogonadal (
hpg
) mouse on
day 1; (c) normal mouse on day 5; (d)
hpg
mouse on day 5; (e) normal mouse on day 20; (f)
hpg
mouse on day 20; (g) normal mouse on day 20; (h)
hpg
mouse on day 20; and (i)
hpg
but by day 20 the effect of gonadotrophin withdrawal is
considerably greater in Leydig cells than in Sertoli cells.
The number of Leydig cells in normal mice did not
change significantly between day 16 of gestation and day 5
after birth, and then increased markedly to 6 ⫻ 10
5
cells per
testis at day 20 after birth. Vergouwen
et al.
(1991, 1993)
showed that labelling of Leydig cells by [
3
H]thymidine was
very low through fetal life from day 14 of gestation, and that
the number of Leydig cells remained constant after birth up
to day 10 before increasing significantly to 5 ⫻ 10
5
cells per
testis at day 18. Together, the results of the present study
and Vergouwen
et al.
(1991, 1993) indicate that after initial
differentiation and proliferation of fetal Leydig cells at about
day 12 of gestation (Gondos, 1980) there is little further
change in the size of the fetal Leydig cell population. In
mice, as in other species, a separate adult population of
Leydig cells arises before puberty with adult cells first
detectable in the mouse about days 7–10 (Baker
et al.
,
1999; Nef
et al
., 2000). Therefore, it is highly likely that the
increase in the number of Leydig cells after day 10 is due to
differentiation and proliferation of the adult population. The
normal number of Leydig cells observed in fetal/neonatal
hpg
testes demonstrates that the number of fetal Leydig cells
is not dependent on gonadotrophins. This finding is in
agreement with studies in sheep in which fetal hypo-
physectomy did not alter the number of fetal Leydig cells
(Hochereau-de Reviers
et al.
, 1995). Currently, little is
known about the factors that govern differentiation and
early proliferation of fetal Leydig cell populations, although
Sry
may be involved in regulating proliferation of the pre-
Leydig cell lineage (Schmahl
et al.
, 2000). Lack of
Wnt-
4
expression appears to be essential for fetal Leydig cell
differentiation (Vainio
et al.
, 1999). Animals lacking Desert
hedgehog (
Dhh
) are feminized at birth, indicating a role for
Dhh
in fetal Leydig cell differentiation or function (Clark
et al.
, 2000).
Unlike the fetal Leydig cell population, failure of normal
numbers of adult Leydig cells to develop after day 5 in
hpg
mice shows that the adult Leydig cell population is
critically dependent on gonadotrophins for establishment of
a normal population size at this time. This finding is
Regulation of numbers of Leydig and Sertoli cells
231
mouse on day 20, in the region of the rete testis. Representative Sertoli cells (S), Leydig cells (L), gonocytes (G), spermatocytes (Sp) and
peritubular cells (P) are labelled, as are lipid droplets (Lp) visible within some Leydig cells. Scale bars represent 10 µm.
0
Leydig cell volume
(µm
3
)
1000
2000
3000
1 5 20 Adult
Age (days after birth)
*
*
Fig. 3. Leydig cell volume during post-natal development in
normal (䊏) and hypogonadal (
hpg
) (䊐) mice. Values are mean ⫾
SEM. *Indicates that there is a significant difference in the number of
cells between normal and
hpg
mice at that age (
P
< 0.05).
0
Cell number per testis
(x10
–4
)
16 18 5 Adult
Day after birth
5
10
15
50
100
150
120
Day of
gestation
*
*
*
*
*
*
0
16 18 5 Adult120
100
200
300
Cell number per testis
(x10
–4
)
0
16 18 51
5
10
15
20
Cell number per testis
(x10
–4
)
(a)
(b)
(c)
Day after birthDay of
gestation
Day after birthDay of
gestation
*
Fig. 4. Changes in numbers of (a) Leydig cells, (b) Sertoli cells and
(c) gonocytes in the testes of normal (䊏) and hypogonadal (
hpg
) (䊐)
mice during development. Values are mean ⫾
SEM (
n=
3–6 mice).
*Indicates that there is a significant difference in the number of
cells between normal and
hpg
mice at that age (
P
< 0.05).
consistent with recent studies on LH receptor knockout
mice which indicate that the numbers of Leydig cells are
reduced in adult animals (Lei
et al
., 2001; Zhang
et al
.,
2001), although the cells were not counted in these studies
and comparisons cannot be made with the results reported
here. Dependence upon gonadotrophins for Leydig cell
proliferation after day 5 correlates with what is known
about control of fetal and adult Leydig cell function.
During fetal development, testosterone concentrations
and expression of steroidogenic enzymes are normal in the
testes of
hpg
mice (O’Shaughnessy
et al.
, 1998). However,
after birth there is a rapid decrease in intratesticular
testosterone concentrations in the testes of
hpg
mice so
that by day 5 testosterone is largely undetectable and
remains undetectable into adulthood (Scott
et al.
, 1990;
O’Shaughnessy
et al
., 1998). Thus, maintenance of fetal
Leydig cell function becomes gonadotrophin-dependent
shortly after birth but as there is little or no Leydig cell
proliferation at this time in normal mice, no effect of
gonadotrophin deficiency on the numbers of cells is
observed.
A study by Ariyaratne
et al.
(2000) has indicated that
initial functional differentiation of adult Leydig cell
precursors in rats might be independent of LH. In mice, as in
rats, this will occur between day 10 and day 20, a period
during which there is little change in the number of Leydig
cells in
hpg
testis. This finding indicates that LH is either
required for proliferation of differentiated adult Leydig cells
during this period or early functional differentiation of
precursors to pre-Leydig cells may be LH-independent,
whereas later development of these Leydig cells is LH-
dependent. Interestingly, in
hpg
mice there is an approxi-
mate doubling of the number of Leydig cells between day
20 and adulthood, about the same percentage increase as in
normal mice. This late increase in the number of Leydig
cells may be partly due to LH-independent differentiation of
Leydig cells in both normal and
hpg
mice or may be due to
induction of Leydig cell proliferation by factors such as anti-
Mullerian hormone and thyroxine (Behringer
et al.
, 1994;
Mendis-Handagama
et al.
, 1998; Racine
et al
., 1998; Teerds
et al
., 1998). In addition, studies on
Dhh
-null mice have
indicated that
Dhh
may be a crucial regulator of adult Leydig
cell and precursor cell differentiation (Nef
et al.
, 2000).
At birth, Leydig cell volumes were normal in the testes of
hpg
mice, which is a further indication that function in these
cells is also normal at this time. Early adult Leydig cell
volume in normal animals was the same as fetal Leydig cell
volume but there was a marked hypertrophy of the cells
after puberty. Leydig cell volume decreased in the
hpg
testes after birth and remained low in the adult mice,
thereby indicating that adult hypertrophy in normal mice is
gonadotrophin-dependent.
The numbers of Sertoli cells in normal mice increased
throughout fetal and neonatal development up to day 20.
This finding is in agreement with a number of earlier studies
in various species which have shown that Sertoli cell
proliferation occurs mainly during fetal and prepubertal life
(Steinberger and Steinberger, 1971; Curtis and Amann,
1981; Orth, 1982, 1984; Johnson
et al.
, 1984; Kluin
et al.
,
1984; Bortolussi
et al.
, 1990; Vergouwen
et al.
, 1991, 1993;
Hochereau-de Reviers
et al.
, 1995). The number of Sertoli
cells determined in the present study at day 20 after birth
and in adult mice (approximately 2.3 ⫻ 10
6
cells per testis)
is in good agreement with the number reported by
Vergouwen
et al.
(1993) after day 18 (approximately 1.8 ⫻
10
6
cells per testis). A number of studies have shown that in
rats a reduction of circulating gonadotrophin
concentrations during the neonatal period leads to a
reduction of about 45% in the number of Sertoli cells (van
den Dungen
et al.
, 1990; Atanassova
et al.
, 1999; Sharpe
et
al.
, 1999, 2000). The results of the present study now
extend this finding to show that, in mice, fetal development
is largely gonadotrophin-independent and that a
requirement for gonadotrophins is not established until late
fetal life. This finding is consistent with earlier studies by
Orth (1984) showing that late decapitation of rat fetuses or
injection of FSH antiserum at day 18 of gestation reduced
[
3
H]thymidine incorporation into Sertoli cells. After birth,
the rate of proliferation of Sertoli cells in the testes of
hpg
mice was reduced significantly, which is consistent with
data described above for neonatal rats and with studies
showing that treatment of intact neonatal rats or neonatal
hpg
mice with FSH will increase the number of Sertoli cells
(Meachem
et al.
, 1996; Singh and Handelsman, 1996).
Interestingly, proliferation continued in the
hpg
testes after
day 20, which may indicate that gonadotrophins are
required to stop Sertoli cell proliferation at the start of
puberty.
The gonadotrophin-independent Sertoli cell proliferation
during fetal life and the continued proliferation after birth in
hpg
mice, albeit at a lower rate, highlights the importance of
other factors in regulating the number of Sertoli cells. Early
proliferation of Sertoli cells, before sex cord formation,
appears to be stimulated by
Sry
(Schmahl
et al.
, 2000) and
there is clear evidence that thyroxine acts to inhibit Sertoli
cell proliferation in the neonatal period (van Haaster
et al.
,
1992, 1993). Regulation of the fetal growth of the number of
Sertoli cells remains unclear, although
Fmr1
, glial cell line-
derived neurotrophic factor and transforming growth factor
α, may all be involved (Slegtenhorst-Eegdeman
et al.
, 1998;
Hu
et al.
, 1999; Levine
et al.
, 2000; Petersen
et al.
, 2000).
The role of these and other factors in determining the final
numbers of Sertoli cells in normal testes remains to be
elucidated.
The testes of
hpg
mice have very low testosterone
concentrations after birth (O’Shaughnessy and Sheffield,
1990; O’Shaughnessy
et al.
, 1998) and the reduced Sertoli
cell proliferation in the neonatal period may be due to lack
of androgen stimulation rather than direct FSH stimulation.
However, Singh and Handelsman (1996) have shown that
neonatal treatment of
hpg
mice with testosterone does not
change subsequent numbers of Sertoli cells in the adult
mice. As testosterone concentrations are normal in
hpg
testes in the fetal period (O’Shaughnessy
et al.
, 1998) it
232
P. J. Baker and P. J. O’Shaughnessy
is likely that the reduced number of Sertoli cells in neonatal
hpg
testes is due directly to lack of FSH.
Primordial germ cells divide mitotically in developing
fetal mouse testes until about day 13.5 of gestation and then
arrest until just after birth when they resume mitosis
(McLaren, 1984). As the number of gonocytes present
during fetal life is determined by day 13.5 it is to be
expected that gonocyte numbers would be normal in
hpg
testes. After birth, gonocytes in both normal and
hpg
testes
re-entered mitosis but from a measure of the number of
germ cells present on day 5 it is clear that the rate of
proliferation is slower in the absence of circulating
gonadotrophins. This may be a reflection of the lower rate
of Sertoli cell proliferation at this time or a decrease in
Sertoli cell activity through loss of FSH and testosterone
stimulation (O’Shaughnessy
et al.
, 1998).
This study was supported by awards from the BBSRC and the
Wellcome Trust. The authors would like to thank W. V. Holt for
assistance in recognition of germ cell morphology.
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Received 25 October 2000.
First decision 7 December 2000.
Resubmitted 15 March 2001.
Accepted 30 April 2001.
234
P. J. Baker and P. J. O’Shaughnessy
