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Morphometric analysis of testicular Leydig cells in normal adult mice.

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THE ANATOMICAL RECORD 204333-339 (1982)
Morphometric Analysis of Testicular Leydig Cells in Normal
Adu It Mice
H. MORI, D. SHIMIZU, R. FUKUNISHI, AND A. KENT CHRISTENSEN
Department of Patholosy and Centml Research Laboomtory,Ehime UniversiCy School of
Medicine, Shigenobu, Ehime, Japan (H.M.,D.S., RE.) and Department of Anatomy and
Cell Biology, The University of Michigan Medical School, Ann Arbor, Michigan 48109
(A.K.C.)
ABSTRACT
Stereological analysis was carried out on Leydig cells in perfusion-fixed testes of normal adult mice.
In a decapsulated testis, the seminiferous tubules occupy 89.3% and the interstitial tissue makes up 10.7% of the volume of the testis parenchyma. The Leydig
cells comprise 3.8% of testicular volume. There are 24.9 million Leydig cells per
cm3 (or gm)of tissue. An average Leydig cell has a volume of 1,533 pm3 and a
surface area of 1150 pm2.
The smooth endoplasmic reticulum (SER) is the most prominent organelle in
the Leydig cells, and has a membrane surface area of 2,428cm2 per cm3 of fresh
testis tissue, which is 8.5 times the surface area of the plasma membrane and
constitutes 56.9% of the total membranes in Leydig cells. Mitochondria occupy
10.1% of the Leydig cell volume or 11.4% of cytoplasmic volume. The inner mitochondrial membrane (including tubular or vesicular cristae) provides a surface
area of about 2855 km2/cell and is 2.26 times that of the outer membrane. There
are approximately 712 cm2 of inner membranes per cm3 tissue. Mouse Leydig
cells have numerous lipid droplets, which average 147 per cell and occupy 5.1%
of the cell volume.
Leydig cells of the mammalian testis secrete
testosterone, necessary to maintain spermatogenesis in the seminiferous tubule and to
regulate male target tissue throughout the body.
The cytoplasm of Leydig cells characteristically contains an abundant smooth endoplasmic reticulum, known to be the site of many
enzymes of testosterone synthesis. Other important steroidogenicenzymes are found on the
inner mitochondria1membrane. Lipid droplets
contain cholesterol esters that may act as substrates for androgen biosynthesis. For a review
of Leydig cell structure and function, see
Christensen (1975).
In correlating structure with function in
Leydig cells, it is important to have accurate
quantitative information on cell numbers and
on the volumes and membrane surface areas
of the various organelles, especially those involved in testosterone synthesis. Modern
methods of morphometry (reviewed by Weibel
and Bolender, 1973)allow such information to
be gained from light and electron micrographs.
We have previously published rather extensive
morphometric studies on Leydig cells in the
testes of normal adult rats (Mori and Chris0003-276X/82/2044-0333$02.50 0 1982 ALAN R. LISS, INC.
tensen, 1980),guinea pigs (Mori et al., 19801,
and elderly men (Mori et al., 1982).In the present study we extend these investigations to the
Leydig cells of normal adult mice. Although
the Leydig cells of several species of mammals
have been the subject of morphometric investigation (see Discussion, and Christensen and
Peacock, 19801,this to our knowledge is the
first such study in the mouse.
MATERIALS AND METHODS
Five young adult male mice (CD-1strain, 78
days old, from Charles River Breeding Labs,
Wilmington, MA) were used after one week of
acclimation. They were maintained on usual
lab chow and water ad libitum.
The right testis was fixed by perfusion
through the thoracic aorta with 3% glutaraldehyde buffered with 0.1 M s-collidine,pH 7.4,
for 15 minutes at room temperature under ether
anesthesia. After perfusion, the testes were cut
into six slices, perpendicular to the long axis
of the testis, and were washed in buffer overnight or longer. Alternating slices were utiReceived July 6, 1982; Accepted Aug 27, 1982.
334
H. MORI ET AL.
lized for light and electron microscopy, making
three slices for each. Subsequent procedures to
prepare the samples for stereology were essentially the same as described previously (Mori
and Christensen, 1980;Christensen and Peacock, 1980).
A detailed account of stereological theory and
practice used in this study can be found in review articles (e.g., Weibel and Bolender, 1973)
and in one of our previous papers (Mori and
Christensen, 1980)."he volume of the interstitial tissue of the testis and the number and
volume of Leyig cells were measured at the
light-microscope level, while the number, volume and surface area of Leydig cell organelles
were analyzed a t the electron-microscopelevel.
Volume densities were determined by pointcounting and were expressed as the volume of
a particular structure per unit volume within
a specified reference space. The stereological
analysis of mouse Leydig cells required four
sampling stages, since the cellular components
exhibited a broad range of size and frequency
(Table 1). In stages I and 11, numerical (Nv)
and volume (Vv) densities were estimated on
light-microscope sections by viewing the specimen through an eyepiece grid containing a
square lattice of 441 points in an area equal
to 1 cm2.Numerical densities were calculated
by means of the Floderus (1944)equation. In
stages 111and IV,numerical and volume densities of Leydig cell organelles were estimated,
using a square, double-lattice test sheet (1:4/
108:432),with an area equal to 442 cm2,placed
over the electron-microscope prints. For the
measurement of surface density (Sv), a coherent multipurpose grid was used, containing
90 test points and 45 test lines of 2.42 cm,
providing a total intersection length of 109 cm.
Stereological analysis produces intrinsic
systematic errors which yield an overestimation or an underestimation of the values, de-
Fig. 1. Low-power light micrograph from a mourn testis
fixed by perfusion with s-collidine-bufferedglutaraldehyde
and embedded in glycol methacrylate. Tissue relationships
are well preserved, providing favorable material for morphometry. x 80.
Fig. 2. Mouse Leydig cells seen in this light micrograph
exhibit a densely stained cytoplasm. Most of the cells contain numerous lipid droplets.Arrows indicate mamphages.
X
1,050.
335
MORPHOMETRY OF TESTICULAR LEYDIG CELLS
TABLE 1. Sampling stages of stereology on mouse Leydig cells
Stage I
Stage I1
Stage I11
Stage IV
Electron microscopy
x 10,250
SV of Leydig cells
N v and Vv of
organelles
60 micrographs
Electron microscopy
~83,200
Sv and Vv of
organelles
Level
Magnification
Density* and
components determined
Light microscopy
x 200
VV of interstitial
tissue
Light microscopy
x 400
Nv and Vv of
Leydig cells
Size per animal
40-60 fields
100 fields
60 micrographs
'Nv, Vv, Sv. Number, volume and surface area per unit volume, respectively
pending on the conditions. These errors have
been corrected according to methods outlined
in detail elsewhere (Mori and Christensen, 1980;
Weibel and Paumgartner, 1978).
RESULTS
TABLE 2. Basic data on mice examined
Animal
No.
1
2
Body
weight
(g)
37
33
Testis weight
Testis volume
(g)
km3)
after perfusion fixation
0.130
0.128
0.125
0.123
The perfusion-fixed testicular tissue of the 3
36
0.128
0.123
-_
present study is well preserved and would thus 4
0.119
33
0.124
0.122
0.127
34
appear to be favorable for stereological anal- 5
ysis. Tissue relationships are well maintained
0.127
0.122
34.6
at the light-microscope level (Figs. 1,2), with Mean
+0.001
10.001
k0.8
fS.E.
little artifactual expansion of intercellular
Fixed testis volume was estimated by dividing the testis weight
space. Organelles viewed in Leydig cells at the by
specific gravity of 1.042 k 0.0008 (N = 8), whereas the
electron microscope level (Figs. 3,4) show no specific gravity for fresh testis was 1.055 f 0.0009 (N= 4).
apparent artifactual changes, and their membranes are clearly visible. The fine structure
was essentially as has been described for mouse
Leydig cells by Christensen and Fawcett (1966).
The testes used in this study weighed 0.127
The smooth endoplasmic reticulum (SER),
2 0.001 gm (mean
SEMI and were estimated to have a volume of 0.122 f 0.001 cm3 an important site of steroidogenic enzymes
(Christensen, 19751, has a volume of 103 pm3
(Table 2).
Our data, listed in Table 3, are expressed in in an average Leydig cell, constituting 6.7%of
terms of three stereological parameters, based the total cellular volume. These values are
on fresh testis (without the capsule): Numer- measurements of the organelle itself, namely,
ical density (N = the number of structures per the volumes of the membrane and the contents,
cm3 of fresh testis), volume density (V = the and do not refer merely to the volume of genvolume of a structure per cm3 of fresh testis), eral areas of the cytoplasm in which SER preand surface density (S = the surface area of a dominates. The surface area of the SER is 2428
structure per cm3of fresh testis). Since one cm3 cm2/cm3tissue. This amounts to a surface area
of fresh testis weighs 1.055 gm,the value per of 9736 p,m2 for the SER of a n average Leydig
cm3 is essentially the same as the value per cell, which is 56.9%of the total membrane surface area in the cell and is 8.5 times greater
gm of fresh testis.
In a decapsulated testis, the testicular tissue than that of the plasma membrane.
Mitochondria are also sites of enzymes imconsists of 89.3% seminiferous tubules and
10.7%interstitial tissue. The Leydig cells oc- portant in testosterone biosynthesis in the
cupy 3.8%of total volume in the decapsulated Leydig cell (Christensen, 1975). The average
testis. One cubic centimeter of mouse testis mitochondrion is 0.43 pm in diameter and 2.72
contains 24.9 f 1.28 million Leydig cells, on pm long. There are 605 mitochondria in a n
the average, which means that a 35-gm mouse average Leydig cell, comprising a volume of
would have about 6 million Leydig cells in both 156 pm3 per cell, which is 10.1% of the cell
volume or 11.4%of the cytoplasm. The surface
testes.
An average Leydig cell has a volume of 1533 area of the outer mitochondria1 membrane is
pm3and a surface area of 1150 pm2.Its nucleus 315 crn2/cm3tissue or 1262 cLm2/cell,while that
has a volume of 161 pm3, constituting 10.5% of the inner membranes (including the tubular
cristae) is 712 cm2/cm3tissue or 2855 pm2/cell.
of the cell volume.
~
*
336
H. MORI ET AL
337
MORPHOMETRY OF TESTICULAR LEYDIG CELLS
TABLE 3.Stereological data on m o u e Leydig cells
Component
Seminiferous tubules
Interstitial tissue
Leydig cells
Parameter'
V
V
N
V
S
Nucleus
Cytoplasm
V
V
Endoplasmic reticulum
Smooth
Rough
Nuclear envelope
Golgi complex
S
Mitochondria
N
V
Outer membr
Inner membr
Peroxisome
Lysosome
s
S
N
V
S
N
V
Multivesicular bodies
S
N
V
Lipid droplets
N
V
Cytoplasmic matrix
V
S
S
Mean value/
cm3 tissue
SEM
(n = 5)
0.8931
0.1069
24.94 x lo6
0.03822
286.82
0.00401
0.03421
0.00379
0.00379
1.28 x los
0.001528
18.700
0.000276
0.001388
0.00258
2428.13
0.00021
222.96
0.00016
38.65
0.00010
64.36
15.09 x 109
0.00388
314.63
712.19
35.96 x 109
0.00025
56.61
4.95 x 109
0.00048
41.89
1.75 x 109
0.00005
9.29
3.66 x 109
0.00194
93.76
0.02456
0.000136
119.80
0.000021
21.000
0.000015
3.304
0.000027
21.074
0.970 x lo9
0.000155
12.287
30.339
2.598 X log
0.000015
4.550
0.187 X log
0.000024
4.031
0.229 X log
0.000004
1.384
0.263 x 109
0.000292
13.411
0.001148
Per average
Leydig cellZ
1533
1150
161
1372
103
9736
9
893
6
155
4
258
605
156
1262
2855
1442
10
227
198
19
168
70
2
37
147
78
376
985
'Dimension of parameters (Note: It is possible to substitute gm approximately for em3 throughout these parameters, since the specific
gravity of mouse testis tissue [ = 1.0551 is near unity): Number (N):No./cm3and No./cell, respectively.Volume (V): cms/cm3and pms/cell,
respectively. Surface area (S):cmz/cm3and pm2/cell,respectively.
a'rhe values per cell are obtained by dividing the value per cm3 tissue by the Leydig cell number per cm' tissue.
The surface areas of the outer and inner mitochondrial membranes constitute 7.4% and
16.7%,respectively, of the total membranes of
the Leydig cells, and thus are 1.1and 2.5 times,
respectively, greater than that of the plasma
membrane. The inner membrane (including
cristae) has a surface area 2.26 times greater
than the outer mitochondria1 membrane.
Fig. 3. Low-power electron micrograph of mouse Leydig
cells, illustrating the quality of preservation of the material
used for morphometry in this study. x 6,300.
Fig. 4. Mouse Leydig cells are characterized by a welldeveloped smooth endoplasmic reticulum (SER), numerous
lipid droplets (Lip), and mitochondria with vesicular and
tubular cristae (Mit). Gol, Golgi complex; Per, peroxisome;
Lys, primary lysosome; Mul, multivesicular body; Gly, glycogen. x 19,500.
The Golgi complex constitutes only 0.3% of
cell volume. Again, this is a measurement of
the organelle itself (membrane plus contents),
whereas a separate count shows that regions
of cytoplasm in which the Golgi complex occurs
constitute approximately 1.9% of cell volume.
Since the Golgi complex is generally included
with the SER in conventional fractionation
centrifugation, we will give some combined
values. The SER plus Golgi complex has a volume of 107 ~m~ per average Leydig cell, constituting 7.0% of the cell volume or 7.8% of
cytoplasmic volume. The combined surface area
of SER plus Golgi is approximately 10,000 Fm2
per cell, which is 8.7 times that of the plasma
membrane or 58.4% of the total membranes of
the average Leydig cell.
The rough endoplasmic reticulum (RER)and
the perinuclear cisternae occupy 0.6% and 0.4%
338
H. MORI ET AL.
of the total cell volume, respectively, and comprise 5.2%and 0.9%of the total membranes of
the average Leydig cell.
Leydig cells in mouse testis have numerous
lipid droplets. In an average Leydig cell, there
are 147 lipid droplets, with a volume of 78 pm3,
constituting 5.1%of the cell volume or 5.7%of
cytoplasmic volume. The average lipid droplet
has a diameter of 1.07 pm and a volume of 0.64
km3.
Peroxisomes or microbodies, identified by the
criteria described in a previous paper (Mori
and Christensen, 1980), have an average diameter of 0.28 pm. An average Leydig cell has
1,442 peroxisomes, which occupy 0.7% of the
cell volume. Each cm3 of testis tissue contains
about 36 billion Leydig peroxisomes.
Lysosomes occupy 1.3%of the cell volume.
About one-third of this volume is comprised of
primary lysosomes, with a n average diameter
of 0.63 pm, while the other two-thirds is made
up by secondary lysosomes (residual bodies and
autophagic vacuoles), with an average diameter of 0.71 pm.
Multivesicular bodies occupy only 0.1% of
the cell volume, and have an average diameter
of 0.41 pm. There are about 70 multivesicular
bodies in the average Leydig cell.
The cytoplasmic ground substance lying between major organelles constitutes 64.2% of
cell volume or 71.8% of the cytoplasmic volume. It contains free ribosomes and polysomes,
microfilaments, microtubules, glycogen, and
other small cytoplasmic components, as well
as soluble materials constituting the “cytosol”
of cell fractionation.
DISCUSSION
The findings of this study provide quantitative information on the number, volume, and
surface area of Leydig cells and their organelles in the testes of normal adult mice. In this
work we have sought absolute values, determined as accurately as we are able by current
stereological methods and necessary corrections. We hope these findings may be useful to
biochemists and other researchers who want
to know the number of Leydig cells per gm of
fresh testis tissue, the surface area of the smooth
endoplasmicreticulum per gm of tissue, or other
values of potential use in biochemical or physiological studies on the testis.
A prominent smooth endoplasmic reticulum
is one of the characteristic features of steroidsecreting cells, and is known to be the site of
a majority of steroidogenic enzymes (see Christensen and Gillim, 1969; and Christensen, 1975,
for reviews).Early observations on Leydig cells
with the electron microscope showed a conspicuous species variation in the abundance of
the SER. Species with a particularly abundant
SER included the guinea pig (Christensen, 1965)
and opossum (Christensen and Fawcett, 19611,
while the hamster (Wing and Lin, 1977) exhibited the least development of this organelle.
Among species with an intermediate complement of SER in the Leydig cells were the mouse
(Christensen and Fawcett, 1966), rat (Christensen and Gillim, 1969), and human (Christensen, 1975). The SER in canine Leydig cells
proved to be highly variable in amount from
cell to cell (Connell and Christensen, 1975).
Zirkin et al. (1980) have confirmed stereologically the relative abundance of SER in the
Leydig cells of the guinea pig, rabbit, dog, rat,
and hamster, and have shown that the amount
of SER is proportional to the level of testosterone secretion per gm of Leydig cells. Their
study did not include the mouse, but it would
be desirable to show how our data on the SER
in mouse Leydig cells relate to their comparative findings in the other species. However,
this comparison is difficult if not impossible
because of differences in the goals of the two
studies. In fact, it is equally difficult to relate
our previous stereological studies on Leydig
cells in rat (Mori and Christensen, 1980),guinea
pig (Mori et al., 1980) and human (Mori et al.,
19821, with the findings reported by Zirkin et
al. (1980), for the same reason. As they point
out in the Discussion of their paper, they do
not claim that their data represent absolute
values, since the findings are uncorrected for
section thickness o r for tissue shrinkage during processing. As a n example of disparity between their findings and ours, they note that
their value for the volume density of the SER
in rat Leydig cells was almost three times as
large as ours (Mori and Christensen, 1980).
That degree of difference might well be expected as a possible result of two effects: First,
the necessary correction for section thickness
(Weibel and Paumgartner, 19781, carried out
in our study but not in theirs, would reduce
their uncorrected value by about half. In addition, the SER in their study may in some
cases have been artifactually swollen, judging
by Figure 3 of their paper. This moderate increase in SER tubule diameter would greatly
increase the measured volume density of that
organelle, since volume varies as the cube of
the diameter. As a result of these differences
in approach between our studies and the paper
of Zirkin et al. (19801, it is difficult for us to
relate their values to our present findings on
mouse Leydig cells or our past studies on these
cells in rat, guinea pig or human.
339
MORPHOMETRY OF TESTICULAR LEYDIG CELLS
TABLE 4 . Surface densities of membranes involved in testosterone synthesis (em2 membrane per em3Leydig cell
cytoplasm)
Species
Smooth ER
membranes
Inner mitochondria1
membranes
(including
cristae)
Reference
Mouse
Aged human
Rat
Guinea pig
70,977
82,984
94,767
102,457
20,818
15,140
27,563
23,515
Present study
Mori et al., 1982
Mori and Christensen, 1980
Mori e t al., 1980
Morphometric data from our present and
previous studies are summarized in Table 4,
comparing the surface areas of membranes of
the SER and inner mitochondrial membranes
(including cristae), which bear most of the enzymes of testosterone biosynthesis in Leydig
cells of mouse, human, rat, and guinea pig.
These are corrected absolute values, and are
expressed in terms of surface area per unit volume of Leydig cell cytoplasm. Since the steroidogenic enzymes are tightly bound to the
membranes, the membrane surface area is a
much better gauge of enzyme concentration than
volume density of the organelles. The value of
the inner mitochondrial membranes, site of enzymes for cholesterol sidechain cleavage, is
similar from species to species, although somewhat lower in the Leydig cell of aged humans.
As expected, the smooth endoplasmic reticulum is most abundant in the guinea pig, although the relative amount is not as much
greater than that of the rat as might have been
expected, especially compared to the value reported by Zirkin et al. (1980). Whether this
relates to methodological differences or is due
to a difference in strains of guinea pigs available in Japan is unclear.
LITERATURE CITED
Christensen, A.K. (1975)Leydig cells. In: Handbook of Physiology. D.W. Hamilton and R.O. Greep, eds. American
Physiological society, Washington, D.C., Section 7,5:57-94.
Christensen, A.K., and D.W. Fawcett (1966) The fine structure of testicular interstitial cells in mice. Am. J. Anat.,
118:551-572.
Christensen, A.K., and S.W. Gillim (1969) The correlation
of fine structure and function in steroid-secreting cells,
with emphasis on those of the gonads. In The Gonads.
K.W. McKerns, ed. Appleton-Century-Crofts, New York,
pp. 415-488.
Christensen, A.K., and K.C. Peacock (1980)Increase in Leydig cell number in testes of adult rats treated chronically
with a n excess of human chorionic gonadotropin. Biol.
Reprod., 22r383391.
Connell, C.J., and A.K. Christensen (1975) The ultrastructure of the canine testicular interstitial tissue. Biol. Reprod., 12:368-382.
Floderus, S. (1944) Untersuchungen uber den Bau der menechlichen Hypophyse n i t besonderer Beriicksichtigung der
quantitativen mikromorphologischen Verhaltnisse. Acta
Pathol. Microbiol. Scand. (Suppl.), 53:l-276.
Mori, H., and A.K. Christensen (1980) Morphometric analysis of Leydig cells in the normal rat testis. J. Cell Biol.,
84:340354.
Mori, H., N. Hiromoto, M. Nakahara, andT. Shiraishi (1982)
Stereological analysis of Leydig cell ultrastructure in aged
humans. J. Clin. Endocrinol. Metab., in press.
Mori, H., D. Shimizu, A. Takeda, Y. Takioka, and R. Fukunishi (1980) Stereological analysis of Leydig cells in
normal guinea pig testis. J. Electron Microsc. (Tokyo),
29:8-21.
Weibel, E.R., and R.P. Bolender (1973) Stereological techniques for electron microscopic morphometry. In: Principles and Techniques of Electron Microscopy. M.A. Hayat,
ed. Van Nostrand Reinhold Co., New York, 3~237-296.
Weibel, E.R., and D. Paumgartner (1978) Integrated stereological and biochemical studies on hepatocytic membranes. 11. Correction ofsection thickness effect on volume
and surface density estimates. J. Cell Biol., 77:584-597.
Wing, T.-Y., and H.-S. Lin (1977) The fine structure of testicular interstitial cells in the adult golden hamster with
special reference to seasonal changes. Cell Tissue Res.,
183:385-393.
Zirkin, B.R., L.L. Ewing, N. Kromann, and R.C. Cochran
(19801 Testosterone secretion by rat, rabbit, guinea pig,
dog, and hamster testes perfused in vitro: Correlation with
Leydig cell ultrastructure. Endocrinology, 107:1867-1874.
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