Alterations of the Fine Structure and Androgen Secretion of the ...

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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