However,. Jones eta!. (1977) were unable to detect any fine structural changes in the. Leydig cells after. 9 weeks of cryptor- chidism. The present study reports.
BIOLOGY
OF
REPRODUCTION
20,
Alterations Interstitial JEFFREY
409-422
of the
Fine
Cells
in the
B. KERR,
Department
Structure
and Androgen
Experimentally
KAThRYN
of Anatomy,
Medical
(1979)
A. RICH’
Monash
Research
Centre,
and
University, Prince
Victoria,
Secretion
Cryptorchid DAVID
M. DE
Clayton,
Henry’s
of the
Rat Testis KRETSER
Victoria
Hospital’.
3168
and
Melbourne,
A ustralia
ABSTRACT The ultrastructural features of the intertubular tissue of the adult rat testis have been studied following the induction of cryptorchidism by surgical translocation of the testes into the abdominal cavity. The principles of stereology were used to assess the changes in the cytological features of the Leydig cells that occur 4 weeks after inducing cryptorchidism. Following the induction of cryptorchidism the principal changes observed were the hypertrophy of the Leydig cells with respect to cellular size and the increased quantities of Leydig cell organdIes, particularly the mitochondria, Golgi membranes and the smooth endoplasmic reticulum. Despite the increase in organdies associated with steroid biosynthesis, serum testosterone levels of cryptorchid animals were either significantly lower or remained unchanged from normal control values, although serum LU levels were consistently elevated following induction of cryptorchidism. Furthermore, stimulation of the cryptorchid testis with hCG resulted in a subnormal increase in serum testosterone levels. The cytological changes in the Leydig cells are suggestive of increased steroid secretion and the normal or lowered levels of testosterone in the peripheral circulation of cryptorchid rats indicate the possibility of a biosynthetic block of testosterone production or alternatively the conversion of testosterone to other metabolites by the seminiferous tubule compartment.
1967,
INTRODUCTION
Although atogenesis elevation
it is well recognized that spermin some mammals is very sensitive to of testis temperature (Steinb#{233}rger
and Steinberger, mentally induced morphology and are
not clearly Previous
testicular experimental reported Altwein 1973;
following cryptorchidism the levels
that elevated
the
enzymes
gen biosynthesis orchid testis
has (Llaurado
Kormano
al.,
1964;
the
a!.,
ventral 1968; 1973).
1971;
associated
with
Skinner
and
have
to of
crypt1963;
suggesting in the
conflicting and
et al., 1977). Amatayakul cells
within
Leydig
the
the than
cells.
have
et
the
7 day
1972;
cryptorchid
more granular (1970) noted cryptorchid reticulum
rat
appearance. that in the 6 rat testis, the of the Leydig
that observed Jones
obtained al.,
microscopical noted that
eta!.
unable to detect any fine structural the Leydig cells after 9 weeks Accepted Received
in
been
Saba
At the light et a!. (1971)
impression usually
However,
changes
Furbeen
by the utilization of In the few limited
1970;
the
state
combining
techniques. have not
results
Leeson,
Gomes,
an alteration
studies
endocrine studies
quantitate
and
serum 1968;
cryptorchid
detailed
Leydig cells techniques.
(Leeson
Hall
evidence no
studies,
cells gave prominent
Rowson,
1971;
have
levels of Eik-Nes,
function
testis exhibited a Leeson and Leeson week experimentally smooth endoplasmic
andro-
investigators
in the 1960;
and previous
structure stereological
Leydig
some
al.,
been
designed
Jones level,
of
prostate weights Free et al., 1969; A decline in the
been noted in the and Dominguez,
the cell
ultrastructural thermore,
Swerdloff, 1974).
impairment by a reduction
et
Despite
induction of the rat have FSH and LH et
decrease (Clegg,
in Leydig
pituitary-
Walsh and Chowdhury,
shown an as measured
in seminal vesicle and (Inano and Tamaoki, Walsh and Swerdloff,
et
in of serum
1972; and
Other studies have androgenic function
of
the
(Amatayakul
and Gittes, Steinberger
activity
of
while
a
Amatayakul 1975).
of experiupon the Leydig cells
there
defined. investigations
axis
became
1972), the effects cryptorchidism function of the
1968)
reported testosterone
it
was more in normal (1977)
were
changes in of cryptor-
chidism.
August 29, 1978. December 30, 1977.
The
409
present
study
reports
the
results
of
a
410
KERR
morphological
analysis
logical evaluation of rat testis following orchidism
and
and
the interstitial the induction
their
relationship
pheral circulating levels Evidence is presented logically
the
but that measured
Leydig
a systematic
cells
become
their elaboration as peripheral serum
of levels
cells of
of the crypt-
the and that
periLU. cyto-
Experiment
testosterone is unaltered
MATERIALS
AND
METHODS
Procedures
Adult male Sprague-Dawley rats (100 days of age) were anaesthetized with ether and the testes were carefully displaced into the abdomen by gently grasping the epididymal fat pad with blunt forceps, care being taken to avoid damage to the testicular artery. Subsequent descent of the testis was prevented by closure of the inguinal canal by sutures. Seventyfour male rats were made bilaterally cryptorchid in this manner and killed at intervals up to 3 months following this procedure. Two separate experimental designs were used.
FIG. 1. Low magnification electronmicrograph normal rat testis, in close proximity to the blood rod-shaped mitochondria (M), smooth endoplasmic
1.
A group
of animals
to the induction of cryptorchidism intervals thereafter for 4 weeks. In was collected from the jugular vein anaesthesia between 0830 and 1000 in this experiment were bled before cryptorchidism and at the time they ultrastructural
hypertrophic,
or decreased.
Surgical
Experiment
stereo-
to
of testosterone to indicate
ET AL.
were
made
studies. 2. Twenty-four rats, experimentally cryptorchid
was
bled
prior
and at 3-4 day this group, blood under light ether h. Other animals the induction of were killed for 100 days of age, and 30 rats of
the same age were used as sham operated controls. At the beginning of the study, a group of 6 intact control animals was killed by decapitation between 08300900 h and blood was collected from each animal. Subsequently, on Days 7, 14, 21 and 28, 6 control and 6 experimentally cryptorchid rats were killed in an identical manner and blood was collected.
Fixation
and Microscopy
At intervals of 5 and 7 days, 4 and 7 weeks and 3 months following the induction of cryptorchidism, 3 animals were selected for perfusion fixation of the testis. Under ether anaesthesia, the testes of these animals were fixed by whole body perfusion via the ascending aorta or by cannulation of the testicular artery. Perfusion fixation of the testis was carried out
illustrating 2 Leydig cells within the interstitial vascular system. The Leydig cell cytoplasm contains reticulum (5) and a Golgi complex (C). X 9,800.
space of the round and
INTERSTITIAL
CELLS
IN CRYPTORCHID
with a mixture of 5% glutaraldehyde, 4% formaldehyde and 0.05% 2, 4, 6-trinitrocresol, buffered to pH 7.4 with 0.2 M sodium cacodylate as previously described by Kerr and de Kretser (1975). Small pieces of testicular tissue were postfixed in osmium tetroxide and following dehydration in graded alcohols, were embedded in a 1:1 mixture of Epon and araldite. For light microscopy, sections 0.5-1 m in thickness were stained with Toluidine blue and photographed with a Leitz Ortholux or Orthoplan microscope using monochromatic light of 550 nm. Ultrastructural observations were recorded from silver or grey thin sections prepared with a Reichert OMU3 ultramicrotome, using glass and diamond knives, stained with lead citrate and examined with a Hitachi flU-liE electron microscope.
Morphometry
of Leydig
Cells
The determination of the volume of Leydig cells of the intact and 4 week experimentally cryptorchid testis was performed at the light microscopical level. Twenty blocks from normal and cryptorchid testes were selected and a 1 im section taken from each was stained and examined with an Orthoplan light microscope. Random intertubular areas containing Leydig cells were photographed at a primary magnification of X 200 and 50 photomicrographs from each group
FIG. 2. Higher magnification contain electron dense granules lamellar (arrowheads) and tubular of smooth endoplasmic reticulum
RAT
were
prepared
area
of
the
TESTIS
at a final whole
cell
411 magnification
and
the
nucleus
of X 1400. of
80
The
Leydig
cells from the normal testes and 76 Leydig cells from the cryptorchid testes selected at random from all micrographs were then individually measured with a desk top planimeter (Keoffel and Easer Co., Germany, 62005). From these data, the average mean area of normal and 4 week experimentally cryptorchid Leydig cells was obtained, from which the average diameter of the nucleus and the whole Leydig cell was calculated, making the assumption that the cells were approximately spherical. The average volume of the nucleus and the cytoplasm was thus determined. For stereological analysis a random selection of 10 blocks from each of 3 animals was taken and thin sections exhibiting a consistent silver color were cut on a Reichert ultramicrotome. The sections were photographed at a primary magnification of X 8000 to include those areas in which Leydig cells contained a nucleus. Twenty micrographs from each group were selected randomly and were enlarged to a final magnification of X 25,500. A systematic stereological analysis of these electron micrographs was performed using the procedures previously described by Weibel et al. (1966). To estimate the volume fraction of a component in the tissue, the area fraction of a random section occupied by the component was measured by
of Leydig cell cytoplasm within within the mitochondrial matrix in shape. The cytoplasm contains (smooth ER) and numerous
the normal testis. Many of the mitochondria (arrows). The mitochondrial cristae are both a moderate supply of interconnected tubules bodies resembling lysosomes (L). X 17,000.
KERR
412 superimposing an overlay points on each micrograph organelles within the cells.
with
1,000 plotted random and scoring hits on the The differential counting of
test points on organdIes provides a measurement of the fractional areas of such components which is proportional to the volume fraction of the component in the tissue (Weibel et al., 1966). By using the independently derived measurement of the average cytoplasmic volume of Leydig cells within normal and experimentally cryptorchid testes as outlined above, the volume fraction obtained at an ultrastructural level was then expressed as the actual volumes (in .Lm3) occupied by the organdIes in the cytoplasm of both groups of Leydig cells.
Hormone Testosterone dose
response
hCG
(hCG). of
50
IU
iv.
to
(Pregnyl, to
normal
human
chorionic
Organon) rats
gonado-
was and
rats
given 4
in a weeks
after surgically induced cryptorchidism. The dose of 50 lU of hCG was chosen from dose response studies in our laboratory and was designed to produce maximal stimulation of testosterone secretion. Blood
:
samples from mm
were
the after
obtained
jugular hCG
under
light
vein immediately administration. All
formed between 1000 and 1200 to clot at room temperature; and stored at -20#{176}Cuntil assayed. Radioimmunoassay. separated and stored measured methods Purified
by double previously rat
LII
at
ether
anaesthesia
prior studies
to and 60 were per-
h. Blood was allowed serum was separated
Serum from -20#{176}Cuntil
antibody described
all assay.
radioimmunoassay (Lee et al.,
(NIAMD-rat-LI-l-II)
was
was was
rats LII
using 1975).
iodinated
with
and used as tracer while the unlabelled preparation was also used as standard. The antiserum used (NIAMD-A-rat-LH 5-1) was raised in rabbits and used ‘
Il
at dilution of 1:37,500. Intra-assay between 3.2 to 8.9% and all samples were included in the same assay.
Analyses
tropin
ET AL.
Testosterone according to
was measured by a previously described
precision ranged from the study radioimmunoassay method (Wang
et
al., 1974). The antiserum was raised against testosterone-3-carboxymethoxime coupled to porcine thyroglobulin. Intra-assay precision was 10.0% with the interassay
precision
being
10.7%.
#{149}
FIG. 3. After 7 days of cryptorchidism, some Leydig cells are seen in mitotic division, exhibiting conspicuous chromosomes (CH) which in this dividing Leydig cell represent the metaphase stage of mitosis. The Leydig cell cytoplasm is densely packed with smooth endoplasmic reticulum and mitochondria (M). A nearby nondividing Leydig cell presents a lobulated nucleus containing a prominent nucleolus. Both Leydig cells and the macrophage are situated within the interstitial space in which scattered collagen fibers (asterisks) are seen. X 4,100.
INTERSTITIAL
CELLS
IN CRYPTORCIJID
RESULTS
Cytological In served apposing ence was
rats, the
the Leydig intertubular
seminiferous
in the evident.
smooth
electron The
cristae
granules
(Fig.
0.4-0.6 membrane
pm
observed in
granular cytoplasm,
closely
clearly
evident
and showed aggregations tubules of
the
the
2)
were
dense inclusions a single finely
noted
in
lysosomal
cytological
within
mitoand
mitochondrial
circular
resembling rats,
filled
bounded by a homogeneous
(Fig.
cryptorchid
cytoplasm cells were
electron
in
Various
diameter, exhibiting
matrix
differ-
or elongated both lamellar
numerous
2).
and
no
reticulum
and
were
matrix
and
lobulated chromatin interconnected
The round demonstrated
tubular
were obbetween
density of their nuclei of Leydig
endoplasmic
cytoplasm. chondria
cells tissue
tubules
ovoid and occasionally peripherally-located (Fig. 1). Many small
In
these
5 days
bodies.
changes of
the
surgery
were and
TESIIS
changes
evident
Observations
normal within
RAT
became
with
chidism, weeks
a
progressively
longer
duration
arrangements
blood-vascular
of
and
intertubular expansion
FIG. nous
4. Some their
complexes
Leydig
cytoplasm. (asterisks).
cells The
in the majority
X 6,600.
7 day of
cryptorchid
other
cytoplasmic
Leydig
lymphatic
cryptor-
became
tubule survey
more
diameter of a
sections which
revealed upon
4 the to
the
of
the
alter, there was an lymphatic sinusoids
marked
decreased. number of
cells elements
tissue did not in the size of the
as the
seminiferous
Light blocks
a number ultrastructural
of
microscopic using 1 pm mitotic analysis
figures were
confirmed to be within Leydig cells (Fig. 3). A most striking change in the cytological features
of
eration
of
which
frequently
tubules cytoplasm,
the
Leydig
cells
smooth
endoplasmic formed
cells of the normal rat. membranes occasionally 15 pm thus occupying
testis
exhibited organdIes
whorls
was
the
enormous are situated
prolif-
reticulum of fenestrated
occurring in compact masses a feature rarely seen in the
in the Leydig
These focal areas rich in attained a diameter of the majority of the
‘
within
more of
reaching a peak after approximately and persisting thereafter. While
general
which
413
..
whorls of smooth membranes peripheral to these membra-
414
KERR
cytoplasmic
volume
fication
these
concentric smooth
(Fig.
whorls
layers endoplasmic
Profiles
of
of
the
4).
At
high
consisted fenestrated reticulum.
Golgi
more evident and were circular configurations,
organdIes
magni-
of
ET AL.
were
cryptorchid
multiple
cisternae
of
normal
(Table
somes
complex usually the
also
became
arranged in large center of which
and
area of following In
sion of cytoplasmic fractional
quently in the
organelle occupied planimetry.
multivesicular of the Golgi
bodies,
in
some
views,
gations
of
small
vesicles
complex. exhibited 10
bodies complex
appeared
in
length
(Fig.
accumulations plasmic reticulum
of
to
derived
The mitochondria extreme elongation
pm
were found and these
7).
from
be
aggre-
the
Golgi
was
In
normal
scattered
areas,
cistcrnae of rough also became apparent
A nalyses
LH.
measured by planimetry, cells from the cryptorchid
significantly
increased
in comparison
(Table 1). The diameter data was in agreement tions sen,
(Christensen 1975; Clark,
diameter mately
of 8.87
increase doubling is
diameter the volume
assumed
that
Both nuclear significantly
and increased.
in cells
substantiated number
of
overlay on as a percentage
FIG.
(Table
the
Part
at levels
one the
of
area mean
of
the
fraction
was each
fractions
cytoplasm
of
the volume 1 obtained by volume of the
is taken of all
into account, the organelles
testosterone testosterone
comparison
1).
values and
of
were
not
to
expressed cell there-
a Leydig
by of
that most
cell
In
9).
Serum
(P
