Tissue & Cell, 1995 27 (2) 129-147

© 1995 Pearson Professional Ltd.

Building a testis
Lonnie D. Russell*, Luiz Renato de Franga t

Abstract, Specific cellular, subcellular and acellular components of the rat testis including the capsule, the
peritubular tissue (tunica propria) and the lymphatic endothelium were analyzed using morphometric tech-
niques at cellular and subcellular levels to yield volume and surface area data. These data were integrated
with previously published data for other cellular components of the rat testis to provide information about
the volumetric composition for virtually every component of this organ. For major cell types (Leydig, Sertoli,
myoid cells and germ cells) the data are expressed to the subcellular level in terms of volume and, in some
instances, surface area. Graphic portrayals of testis constituents are used for rapid visual understanding of
testis structure. The data presented herein are useful in conjunction with biochemical data to describe
physiological properties of cells and cell components and also for understanding how structure differs under
experimental and in pathological situations.

Keyvvords: Morphometry,testis, rat, electron microscopy, organelles

Introduction 1988; Russell et al., 1994; Wrobel and Schimmel, 1989;

The testis is a complex organ, producing both sperm
and androgens that serve to maintain reproductive func-
tion and secondary sex characteristics. The germ cells
form sperm in the process of spermatogenesis whereas
the somatic cells of the testis are in support of both of
these functions. The structural components of the three
major testicular somatic cells, the Leydig cell (Mori and
Christensen, 1980; Russell et al., 1992; Sinha Hikim
et al., 1989a; Zirkin et al., 1980), the Sertoli cell (Ghosh
et al., 1991, 1992a,b; Sinha Hikim et al., 1989b; Ye
et al., 1993), and the peritubular cell (Kurohmaru et al.,
1990) have been quantified to the subcellular level.
From these studies insights into function of testicular
somatic cells have been gained by relating various
structural parameters to functional parameters. Most
morphometry has been conducted for the rat although
morphometric studies of other species are available
(Kurohmaru et al., 1990; Mendis-Handagama et al.,
* Laboratory of Structural Biology, Department of Physiology, Southern Illinois
University, School of Medicine, Carbondale, Illinois 62901-6512, USA.
Department of Morphology, Institute of Biological Sciences, Federal University
of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil 31270-901 CP 2466.
* This work was supported in part by a fellowship awarded to Luiz Renato de
Franga from the Brazilian Research Council (CNPq).
Sinha Hikim et al., 1988, 1989a, b, 1991, 1993).
Only recently has the population of germ cells been
subjected to morphometric analysis at both the light
(Franga et al., 1995b; Wing and Christensen, 1982) and
electron microscope levels (Franga et al., 1993, 1995b)
to determine the structural evolution of germ cells during
the long process of spermatogenesis. There are, however,
data missing for a few testis components of the rat.
These include a determination of the quantitative fea-
tures of the testis capsule, the peritubular layer (tunica
propria) and related extracellular material and the vascu-
lar cells. It is one of the objectives of this report to fill
in the gaps in volumetric and surface area determinations
for testis components. With complete data it would be
possible to construct or to build a rat testis, for the
most part from its subcellular components. Such an
analysis would greatly facilitate an understanding of
testis structure and would serve as a basis from which
to relate functional parameters and to understand the
impact of experimental protocols and pathology. It is
therefore our second objective to construct a testis from
its components based on morphometric data presented
in this report and data published elsewhere.
Methods

Received I October 1994 Animals and tissue preparation

Accepted 17 November 1994 The rats used in the present study were those utilized in
Correspondence to: Dr Lonnie D. Russell a previous morphometric study (Ye et al., 1993). The
 130 RUSSELL AND DE FRANqA

Table I Seminiferous tubule tunica propria volume density (Vv%) details of animals utilized and tissue preparation are
parameters (mean-+ S.E.) in the rat
presented in that paper.
Extracellular matrix 34.9 _+0.6
Myoid cell 42.1 _+1.2 Morphometry
Clear cell 2.1 + 0.8
Endothelial cell 20.9 _+1.7 Four rats were utilized to obtain all parameters in the
present study. The general plan for morphometry of the
* Tunica propria comprises 1.2% of the testis. testis and for determination of the volumetric and
surface area parameters of its components followed
previously described procedures (Sinha Hikim et al.,
Table 2 Myoid cell volume 6tm 3) and volume density (Vv%) 1988, 1989a, b) which provided absolute measurements
parameters (mean _+S.E.) of cell constituents.
Volume Volume density
Peritubular layer. Montages made from negatives taken
Cell 807 _+124 at 9000 times magnification and enlarged to 22 100 times
Nucleus 116+_14 14.4_+1.5 original size of round or near round tubular profiles
Cytoplasm 691 4-113 85.6 -+ 1.5
Ground substance 606 __ 108 75.1 -+3.4 were constructed from individual electron micrographs
Mitochondria 27.3 -+4.2 3.4 _+0.4 to provide proportional sampling (Ye et al., 1993) of
Smooth endoplasmic reticulum* 31.6_+ 1.9 3.9_+0.5 the peritubular cell layer.
Rough endoplasmic reticulum 17.7 _+1.8 2.2 4- 0.5
Golgi and associated vesicles 4.7 + 2.2 0.6 4- 0.3 Initially, the volumes of myoid cell nuclei were deter-
Lyosome 2.3 _ 1.4 0.3 4- 0.1 mined by light microscope serial section reconstruction
Multivesicular body 1.2 _+0.8 0.15 -+0.1 (x 1250) of five nuclei from each animal used in the
Lipid 0+ 0 --
study. Serial sections (1.38 pm in thickness) of nuclei
* Includes micropinocytotic vesicles within the cytoplasm. were performed on an MT-1 microtome (Porter Blum,

A Major Structural B Subdivisions of Structural

Components* Components

Intertubutar Space Lymphatic

Space
7.7% 6.6%

Peritubular Tissue 1.2%

Seminiferous
Seminiferous Tubule Epithelium
& Rete Testis
85.8%

6.5% Oapsule .,~

*numbers from Ghosh et al (1992) and Russell et al. (1992) were adjusted for a capsule Vv% of 6.5%
(Johnson and Neaves, 1983)

**considered here as part of the seminiferous tubule

Fig. 1 ( h ) The major structural components of the testis (oval structure); (B) subdivisions of those components. The volume occupancy (volume
density percentage) of the components is indicated.
 BUILDING A TESTIS 131

" Vessel I Vessel "

Wall 0.55% [ Lumen 0.55%

Lymphatic Space***
3.7%
2.2%

Myoia I E. othe,a, Extracellular Space

Cell 0.50% n ~o~^~ Cell 0.25% & Matrix 0.42%

Sertoli Germ
Cell Cell
16.2% 55.9%

i
Tubular
Lumen
12.5%

Rete ?

Fibroblasts & Smooth Muscle

1.5%
Space

***data with this type of breakdownof the interstitial space are from Mendis-Handagama et al. (1988)

Fig. 2 The testis shown in Figure 1B is subdivided yet again to show the volumetric composition of the cell types comprising this organ.

S o r v a l l ) a c c o r d i n g to the m e t h o d described b y
Table 3 Myoid cell surface area (gin2) and surface density (Sv) K u r o m a r u et al. (1990). T h e v o l u m e o f m y o i d cell was
parameters (mean ± S.E.) d e t e r m i n e d b y e m p l o y i n g the following f o r m u l a to p o i n t
c o u n t i n g data:
Surface area Surface density
M y o i d cell v o l u m e =
Plasma membrane 5628 +_1046 6.88 ± 0.23
Nuclear membrane 407 +46 0.53 ___0.09 p o i n t s over cell
Outer Mitochondrial membrane 443 _+96 0.54 ± 0.03 x nuclear v o l u m e
Inner Mitochondrial membrane 605 ± 118 0.74 ± 0.03 p o i n t s over nucleus
Smooth endoplasmic reticulum* 1261 ± 276 1.54 ___0.15
Rough endoplasmic reticulum 821 ± 112 1.04 ± 0.09 where the p o i n t s for the counts were o b t a i n e d b y super
Golgi and associated vesicles 163 ± 65 0.23 ± 0.09 i m p o s i n g a t r a n s p a r e n t o v e r l a y grid ( m u l t i p u r p o s e g r i d )
Lysosome 73±12 0.10±0.02 o n electron m i c r o g r a p h s ( x 5 9 0 0 ) .
Multivesicular body 10 ± 5 0.02 _+_0.02
The v o l u m e densities o f m y o i d cell organelles was
* Includes micropinocytoticvesicles within the cytoplasm. d e t e r m i n e d in high ( x 22 100) m a g n i f i c a t i o n m o n t a g e s
132 RUSSELLAND DE FRAN(yA

T~ble 4 Testis components

Component Volume density Volume Number/testis Reference

(Vv%) (gm 3) ( X 10 6)

Capsule 6.2 0.110 Johnson et al., 1980

1.9 0.030 Mori and Christensen, 1980
6.5 0.110 Johnson and Neaves, 1983
10.1 0.180 Johnson et al., 1984
Intertubular space 9.7 Roosen-Runge, 1955
15.7-16" 0.241 Mori and Christensen, 1980
11.4 0.141 Kerr et al., 1985
9.45 Mendis-Handagama et al., 1988
8.0 0.150 Keeney and Ewing, 1990
8.2 0.130 Ghosh et al., 1992a; Russell et al., 1992
6.5 0.102 Russell et al., 1993
Blood vessels 1.63 Mendis-Handagama et al., 1988
1.2 0.020 Ghosh et al., 1992a; Russell et al., 1992
1.9 0.030 Corbin, 1993
1.2 Present report
Vascular wall 0.6 Present report
Vascular lumen 0.6 Present report
Lymphatic space b 4.44 Mendis-Handagama et al., 1988
Connective tissue elements 0.61 Mendis-Handagama et al., 1988
Macrophages 0.3 0.003 Kerr et al., 1985
8.0 Kerr et al., 1987
0.23 477 gm3 (one cell) Mendis-Handagama et al., 1988
7.5 (56-day-old) Hardy et al., 1989
0.6 0.010 Ghosh et aL, 1992a; Russell et al., 1992
Leydig cell 1.7 Roosen-Runge, 1955
2.7-2.8 ~ 0.041 Mori and Christensen, 1980
2.2 Ewing et al., 1983
3.0 0.037 Kerr et al., 1985
2.2 Zirkin and Ewing, 1987
2.55 Mendis-Handagama et al., 1988
3.4 0.065 Keeney and Ewing, 1990
2.4 0.038 Ghosh et al., 1992a; Russell et al., 1992
Seminiferous tubules 90.3 Roosen-Runge, 1955
82.9 Johnson et al., 1980
82.4-84.0" 1.269 Mori and Christensen, 1980
88.6 1.099 Kerr et al., 1985
90.2 1.374 Kerr, 1988a
90.45 Mendis-Handagama et al., 1988
92.0 1.720 Keeney and Ewing, 1990
91.8 1.400 Ghosh et al., 1992a; Russell et al., 1992
93.5 1.450 Russell et al., 1993
Peritubular tissue 1.27 Present report
Intercellular space & matrix 0.44 Present report
Myoid cells 6.1 (56-day-old) Hardy et al., 1989
0.53 Present report
Clear cells (monocyte) 0.03 Present report
Endothelial cells 0.27 Present report
Seminiferous epithelium 64.4 Johnson et al., 1980
81.1 Russell et al., 1990
78.4 1.200 Ghosh et al., 1992a; Russell et al., 1992
78.0 1.210 Russell et al., 1993
Tubular lumen 13.4 0.213 Ghosh et al., 1992a; Russell et al., 1992
15.5 0.243 Russell et al., 1993

a Not including testis capsule.

b Definition of lymphatic space does not include cells and extracellular matrix.

b y p o i n t c o u n t i n g m e t h o d o l o g y . O v e r 1000 p o i n t s were the m y o i d cell p l a s m a m e m b r a n e a n d o r g a n e l l e m e m -

u s e d to s a m p l e the p e r i t u b u l a r layer i n each a n i m a l .
T h e v o l u m e o f each o r g a n e l l e was d e t e r m i n e d as the
p r o d u c t o f the cell v o l u m e a n d the v o l u m e d e n s i t y o f a
p a r t i c u l a r organelle.
M o r p h o m e t r y for surface a r e a m e a s u r e m e n t s was
c o n d u c t e d u s i n g a M e r t z grid o v e r l a y o n m o n t a g e s
f o l l o w i n g s t a n d a r d stereological m e t h o d s ( W e i b e l a n d
B o l e n d e r , 1973). To m a k e surface a r e a d e t e r m i n a t i o n s
b o t h p o i n t s o v e r the m y o i d cell a n d i n t e r s e c t i o n s w i t h
b r a n e s were c o u n t e d a n d recorded. T h e m y o i d cell
p l a s m a m e m b r a n e was c o n s i d e r e d to i n c l u d e p i n o -
cytotic vesicles.
T h e surface d e n s i t y ( S v ) o f p a r t i c u l a r m e m b r a n e s was
d e t e r m i n e d u s i n g the f o l l o w i n g f o r m u l a :
2I
Sv- pt z
w h e r e I is the n u m b e r o f i n t e r s e c t i o n s o n the m y o i d cell

............ i ......................... ........... i ............
I IMV V VI VII
STAGE
Fig. 3 The volumetric composition of the seminiferous epithelium is depicted throughout nine subdivisions of the cycle (roman numerals are
stages) of the seminiferous epithelium. (From Biology of Reproduction 49:1215-1228, 1993 with permission of the Publisher).
plasma membrane and organelle membranes and Pt is
the number of points over the myoid cell and Z is the
length of the line between points in terms of the magnifi-
cation of the micrograph.
To determine surface (S) area from surface density,
the following formula was applied:
S = SvV
where V equals the volume of the myoid cell.
Testis capsule. A square lattice was placed systematically
over electron micrographs taken at x 1500 and enlarged
to x 3900 and the number of points over cells and
extracellular material were determined. The number of
points used for each animal was approximately 4600.
Blood vessels and peritubular tissue. A square lattice
containing 441 points was placed over tissue at x 1250
magnification. Points lying over vessel walls, vessel
lumina and peritubular tissue (tunica propria) were
recorded. About 50 000 total points were recorded for
each animal.
BUILDING A TESTIS 133
............ i ............ i' ......................i ,:- 3s,oo0
o
e-
_=.
o
"c
VIII IX-XI XII-XIII XIV
Results
In the present study data were obtained for testis
components that had not previously been analyzed
morphometrically. These were the peritubular region,
the vascular wall and vascular lumen and the testis
capsule. Data for the rete testis has not been determined
for the rat by any author to our knowledge. Such data
are difficult to obtain given the regional concentration
of the rete in the rat testis.
Morphometric data for the testis capsule demonstrate
that 77.3% of the capsule is collagen, filling the extra-
cellular space, and 22.7% is fibroblastic elements.
Smooth muscle elements were not observed in the ran-
domly selected micrographs used in our preparations.
The blood vessels comprise 1.2% of the testis. The
compartment is evenly divided between vascular wall
(0.6%) and vascular lumen (0.6%).
Morphometry of the peritubular layer or tunica pro-
pria (Table 1) shows that ceils comprise 65.1% of the
peritubular layer (0.78% of the testis), and extracellular
space comprises 34.9% of this layer (0.42% of the testis).
Of the cell types present, myoid cells average 807 gm3
134 RUSSELL AND DE FRAN(yA

Table 5 Leydig cell volttme density (Vv%), volume (grn3), surface area (~tm2) and number

Component Volume Volume Surface area Leydig cell Reference

density (%) Number/Testis (x 106)

Cell 1209 1517 33.6 Mori and Christensen, 1980

1640 21 Ewing et al., 1983
28 Kerr et al., 1987
2200 18.5 Zirkin and Ewing, 1987
2255 22.2 Keeney et al., 1988
1167 Mendis-Handagama et al., 1988
26 (56-day-old) Hardy et al., 1989
21.7 (60-day-old) Bortolussi et al., 1990
2405 26.7 Keeney and Ewing, 1990
1850 Mendis-Handagama et al., 1990b
1378 1550 28 Russell et al., 1992
1197 2888 Russell et al., 1993
Nucleus 12.4 150 149 Mori and Christensen, 1980
11.9 138.6 Mendis-Handagama et al., 1988
15.6 204.8 194 Russell et al., 1992
14.8 177 354 Russell et al., 1993
Nucleolus 2.5/nudeus 5.1 Russell et al., 1992
Cytoplasm 87.6 1059 Moil and Christensen, 1980
88.1 1028 Mendis-Handagama et al., 1988
84.4 1173 Russell et al., 1992
Cytoplasm ground substance 61.9 748 Mori and Christensen, 1980
26.7 311.2 Mendis-Handagama et al., 1988
40.8 562 Russell et al., 1992
Smoothendoplasmic reticulum 11.4 ~ 138a 10458 ~ Moil and Christensen, 1980
32.8 b Zirkin et al., 1980
14000 Ewing et al., 1983
19000 Zirkin and Ewing, 1987
40.1 468.3 69920 Mendis-Handagama et al., 1988
750 Mendis-Handagama et al., 1990b
17.3 c 237.9 30 538 Russell et al., 1992
9976 Russell et al., t993
Mitochondria 11.3 137 Mori and Christensen, 1980
19.7 b Zirkin et al., 1980
12.9 150.9 Mendis-Handagama et al., 1988
210 Mendis-Handagama et al., 1990b
12.0 ° 165.8 Russell et al., 1992
Outer mitochondrial membrane 1641 Mori and Christensen, 1980
2900 Ewing et al., 1983
5200 Zirkin and Ewing, 1987
11 507 Mendis-Handagama et al., 1988
2740 Russell et al., 1992
4456 Russell et al., 1993
Inner mitochondrial membrane 2920 Mori and Christensen, 1980
4900 Ewing et al., 1993
8900 Zirkin and Ewing, 1987
35294 Mendis-Handagama et al., 1988
6748 Russell et al., 1992
6810 Russell et al., 1993
Rough' endoplasmic reticulum 0.7 9 539 Mori and Christensen, 1980
30.1 b Zirkin et al., 1980
700 Ewing et al., 1983
1 11.8 6972 Mendis-Handagama et al., 1988
4.6 c 63.9 5679 Russell et al., 1992
Gotgi 1.4° 17 Mori and Christensen, 1980
3.2 37.5 5368 Mendis-Handagama et al., 1988
0.7 ° 10 525 Russell et al., 1992
Lysosomes 0.6 7 Mori and Christensen, 1980
1.1 13.3 Mendis-Handagama et al., 1988
0.4 c 5 Russell et al., 1992
0.4 4.5 Russell et al., 1993
Multivesicular body 0.3 3 Mori and Christensen, 1980
0.04 0.5 Mendis-Handagama et al., 1988
Peroxisomes 1.2 15 Mori and Christensen, 1980
0.34 4 Mendis-Handagama et al., 1988
0.6 11 Mendis-Handagama et al., 1990b
1.5 c 20 Russell et al., 1992
1.5 17.9 Russell et al., 1993
Lipid 0.2 3 Mori and Christensen, 1980
0.3 b Zirkin et al., 1980
0.8 Zirkin and Ewing, 1987
 BUILDING A TESTIS 135

Table 5 (continued)
Component Volume Volume Surface area Leydig cell Reference
density (%) Number/Testis ( × 106)

0.7 7.8 Mendis-Handagama et al., 1988

0.05 ° 0.7 Russell et al., 1992
0.13 1.6 Russell et al., 1993

a Includes Golgi.
b Expressed per cytoplasm of cell since cell volume was not given.
Recalculated to be expressed per cell.

95%

90%

85%

80%

75%

70%

65%

Round 60%
I,u Spermatids
0
55%
Z
uJ
0
E 50%
Ill
a.
>-
I-- 45%
Z
UJ 40%
Q
:S
35%
,,J
0
>
30%

25%

20%

15%

10°/, 10%

5%

0%

I IMV V VI VII VIII IX-Xl XlI-XlII XlV

STAG E

Fig. 4 The percentage composition of the seminiferous epithelium at nine designated stage groupings (Roman numerals are stages) during the
cycle of the seminiferous epithelium.

in volume and comprise 42.1% of the volume of the
peritubular layer (64.7% of the cells) (Tables 1 & 2).
The endothelium of the lymphatic system comprises
20.9% of this layer (32.1% of the cells). The 'clear cell'
(Hermo and Clermont, 1976) or most probably mono-
cyte comprises 2.1% of the volume of the peritubular
cell layer (3.2% of the cells in the tunica propria).
The subcetlular composition of the myoid celt
indicates that the nucleus, 116 gm 3 in volume, occupies
about 14.4% of the cell with the remaining 85.6% of
136 RUSSELL AND DE FRANGA

the cell being cytoplasm. The organelle composition of
the cell is shown in Table 2. The surface area of the
myoid cell, including pinocytotic vesicles was determined
to be 5628 gm 2 (Table 3).
Discussion
The data provided in this report represent a small
portion of the total work presented, to date, on the
structural composition of the testis. However, these
data, with one exception, complete the analysis of the
various components of the testis. That exception is the
rete testis, whose volumetric composition is difficult to
analyze due to sampling considerations. In the rat where
the rete is small, we estimate the rete testis to comprise
less than 1% of the entire testis although accurate data
are yet to be obtained. In this report, rete testis data
are included with that of the seminiferous tubules. Thus
with completion of data on the volumetric composition
of the testis it is now possible to 'build' a testis.
The volumetric composition of the testis is provided
in Table 4. Data presented in this table were obtained
from a variety of sources including our own to show
the spectrum of values that have been obtained. In
addition to values which are volumetric or three-
dimensional, the volume density (Vv%) or percentage
occupancy for each component is also indicated (Figs 1
&2).
The data chosen for utilization in Figures 1 and 2 are
primarily from this laboratory given that those data are
the most comprehensive source of data for the testis as
a whole. However, to fill in gaps data from other sources
are utilized. Since data are expressed in various ways it
was necessary to recalculate some data from the original
source to fit into the scheme presented. For example,
some data are expressed per testis, per parenchyma, per
cytoplasm and per cell. In this paper they were recalcu-

A In In B B P! PI L Z P P P P P P P P P-Di S
100

23-- J
22--
21--
20_
19--
18-
17_

16--
15-
14
13_

•~ 12-
11_
10_

38- ! 9_

7--
6--
MITOCHONDRIA

5--
4-
3--
2-

8 0.Ts- GOLGI
~ 0.50-
0.25 --
0
I-XIV I n-Iv V VI VII VIH IX-XI XII-XIH XIV I II-IV V VI VII VIII IX-XI x n - x n I x I v

Fig. 5 The percentage'volume occupancy of the components of spermatogonia and spermatocytes at nine designated stage groupings ( R o m a n
numerals are stages) during the cycle of the seminiferous epithelimn. The graph shows the volume of most components in size relationship to their
actual volume, however, the percentage volume occupancy of the ground substance and the nucleus are simply given at the top of the Figure.
(A = A spermatogonia; In = intermediate spermatogonia; B = B spermatogonia; P1 = preleptotene spermat0cytes; L = leptotene spermatocytes;
Z = zygotene spermatocytes; P = pachytene spermatocytes; Di = diplotene spermatocytes; S = secondary spermatocytes).
 BUILDING A TESTIS 137

Table 6 Sertoli cell volume density (Vv%), volume (~n3), surface area (~tm2) and mtmber

Component Volume Volume Surface area Sertoli cell Reference

density Number/testis ( x 106)

Cell 9.9-13.6 b 2000-3200 Roosen-Runge, 1955

18-18.2 Bustos-Obregon, 1970
43.8 de Jong and Sharpe, 1977
11.3 Johnson et al., 1980
21.9 Wing and Christensen, 1982
6012 a Wong and Russell, 1983
12163 a Weber et al., 1983
660 Morales, 1984
24.0 34.0 b'° Bugge and P16en, 1986
19.9-28.6 b 5300-8000 41.4 Kerr, 1988a
8000-15000 Kerr, 1988b
15.5 Orth et al., 1988
45.9-53.0 d Russell et al., 1988
35.3-40.1 Wang et al., 1989
31.1-33.2 Berndtson and Thompson, 1990
30.0 Bortolussi et al., 1990
16.6-18.9 ° 5034-5069 ° Russell et al., 1990
5362 20085 40.5 Ghosh et al., 1992a
62.0 Van Haaster et al., 1992
11 913-15 804 Franga et al., 1993
33.6 Hess et al., 1993
17 23.5 b 5368-7822 11 701-16224 Ye et al., 1993
40.0-41.3 Fran~a et al., 1995a
19.4-26.7 b Fran~a (data not published)
Nucleus 15-23 1140-1260 Kerr, 1988b
525-592 Russell et al., 1988
558-680 Wang et al., 1989
10.7-11.1 542-559 Russell et al., 1990
14.9 77O Ghosh et al., 1992a
5.8-8.3 401 477 257-553 Ye et al., 1993
Cytoplasm 88.9-89.3 4475-4527 Russell et al., 1990
85.1 4592 Ghosh et al., 1992a
91.7-94.2 4923-7366 Ye et al., 1993
Mitochondria 8.0-9.5 500-650 Kerr, 1988b
7.5-9.5 f Ueno and Mori, 1990
5.1 g 272.9 Ghosh et al., 1992a
4.2--6.0 245-389 Ye et al., 1993
Outer mitochondrial membrane 3098 Ghosh et al., 1992a
1685-2680 Ye et al., 193
Inner mkochondrial membrane 8937 Ghosh et al., 1992a
7705-11 360 Ye et al., 1993
Smooth endoplasmic reticulum 9-21 450-1700 10500-22000 Kerr, 1988b
6.3 g 337 26 124 Ghosh et al., 1992a
12.2-16.9 745-1112 11014-19762 Ye et al., 1993
Rough endoplasmic reticulum 0.3-1.5 20-80 800-3500 Kerr, 1988b
1.0 g 55.6 5681 Ghosh et al., 1992a
0.02-1.1 1-67 590-2211 Ye et al., 1993
Golgi 0.35-0.88 20-65 1400-1900 Kerr, 1988b
0,35 g 18.9 1205 Ghosh et al., 1992a
0.4-0.9 28-58 877-2353 Ye et al., 1993
Lysosomes 1.6-2.6 80-200 Kerr, 1988b
1.3 g 69.3 Ghosh et al., 1992a
1.6-2.8 83-220 Ye et al., 1993
Multivesicular bodies 0.06 g 3.3 Ghosh et al., 1992a
0-0.1 0-6 Ye et al., 1993
Lipid 0.4-2.5 3.5-29 Kerr et al., 1984
1-7 ~ Ueno and Mori, 1990
0.5-g 27.4 Ghosh et al., 1992a
0.3-3.0 14-235 ¥ e et al., 1993
Ectoplasmic specialization 1.1 g 59.7 3345 Ghosh et al., 1992a
655-1518 Franga et al., 1993
672-1664 Ye et al., 1993

a Only at stage V.
b Within the seminiferous epithelium.
° Only 6 stages were considered.
d Stage V and VI.
Stage I-III and VII-VIII.
f Counts were made only in the basal compartment.
g Recalculated to be expressed per cell.
138 RUSSELL AND DE FRAN(~A

Table 7 Volumes(~rn3) of individual germ cells (mean_+S.E.)

Stages Spermatogonia/primary Primary/secondary Round/elongate
spermatids Elongate spermatids
spermatocytes spermatocytes
I 492+30 (Type A I-XIV) 658+ 135 (P) 1279+86 (Step 1) 1198___ 100 (Step 15)
II-IV 433 _ 40 (In I-IV) 1112_+ 149 (P) 1443 _ 38 (Step 2-4) 1262 _ 80 (Step 16-17)
V 352___23 (Type B V-VI) 1541 + 134 (P) 1690_+ 169 (Step 5) 1105 _ 172 (Step 17
VI (see Stage V) 1778 _+172 (P) 1901 _+142 (Step 6) 899 _+81 (Step 18)
VII 332_+ 37 (P1) 2498 _ 118 (P) 1914_+73 (Step 7) 580_+ 115 (Step 19)
VIII 468 _ 50 (P1) 2816 _+223 (P) 1513 _+116 (Step 8) 452 + 106 (Step 19)
IX-XI 574 _ 36 (L) 2624 _+225 (P) 1277 _+31 (Step 9-11 )
XII-XIII 497+40 (Z) 4202_+ 123 (P/Di) 1164_+ 138 (Step 12-13)
XIV 766 _+59 (P) 2507 _+220 (S) 1189 _+97 (Step 14)

steps 1 2-4 5 6 7 8 9-11 12-13 14 15 16-17 17 18 19 19

37.0 -
~35.$
34.0 .[ . . . . . I

33.$
31.0
29.$
28.0
26.$
25.0
23.$
22.0
20.5
19.0 ................. i ......................
17.5 . . . . . . . . I......... I .......................
Endoplasmic Reticulum
16.0
14.5 J
13,0
0
11.5
10.o
8.5
7.0
5.$
4.0
Z.$
1.0
• Aerosome
0.75
0.50.
0.25 -
0.00.
I H-IV V VI • Vll VIH IX-XI XII-XHI XIV I II-IV V VI VII VIH

Fig. 6 The percentage volume occupancy of the components of spermatids (indicated as steps) at nine time periods (Roman numerals) during the
cycle of the seminiferous epithelium. The graph shows thelvolume of most components in relationship to their actual volume however the
percentage volume occupancy of the ground substance an d the nucleus are simply given 'at the top of the figure.

lated in a uniform manner to be expressed: eithe r per parameters, volumetric data is equally meaningful or
testis or per cell. Thus, the data utilized may not be more meaningful than surface area data, however, sur-
recognizable from the original source. For the major face area data are more useful in some instances that
cell types of the testis, Leydig cells, Sertoli cells and relate to physiologic responses. For example, the surface
germ cells the data are presented at the subcellular level area of membranes such a smooth endoplasmic reticu-
(Tables 5, 6 and 8-11, respectively). Although the focus lum or inner mitochondrial membrane may relate to the
of this report is on three-dimensional information, we enzymatic capabilities of these membranes during steroid
also include surface area determinations. For some cell synthesis or metabolism. We have also provided surface
 BUILDING A TESTIS 139

A
D
L
U
M
I
N
A

I : II-IV V VI VII VIII IX-Xl XII-XIII XIV

STAGE

Fig. 7 Diagrammatic representafio~ of the structures related to the surface of the Sertoli cell at nine designated stage groupings during the cycle o f
the seminiferous epithelium. The ~op of the graph is considered to be 100% of the surface of the Sertoli cell. (From Biology of Reproduction 49:
1215-1228, 1993 with permission of the Publisher).

area data when such ~measurements are thought to
contribute to the overall understanding of testis
composition.
Numbers, by themselves, do not lead to an adequate
conceptualization of testis composition or at least do
not provide a rapid visual grasp of testis makeup. To
facilitate rapid understanding of testis structure, we
have also prepared Figures (Figs 3-7) to show testis
composition.
From examination of similar parameters it is obvious
that there is considerable interlaboratory and intra-
laboratory variability in the data for any one parameter
in a normal animal. In some instances there are differ-
ences that are more than 10-fold from one laboratory
140 RUSSELL A N D DE FRAN(~A

+1 +1 +1 +1 +1 +1 +1 ~1

?
+l+l+l+l+l+l÷l+l÷l+l+l÷l+l+l÷l+l+l÷l+l+t+l+l+l+l

r~

~1 ~i ÷l ÷l +l ÷l ÷l +l ,÷l +l ÷l ÷l +~ +l

+~
+l+i+i+l+i+l+l+l+l÷l÷l+l÷l+l+l÷l+l÷l÷l+l÷l+l+l+t+l+l+I+l+l+l+l+l+l+l

+i+l+l+l+l+i+l+l+l+l+l+l÷l+l+l÷l+l÷l÷l+l÷l+l+l÷l+l+l+l+l+l+l+l+l+l+l
2
.a
0'3

4-1 4-1 4-] -I-t 4-1 4-1 4-1 -I-i 4-1 4-1 4-1 4-1 4-1 4-1 4-1 4-1 4-1 4-1 4-1 4-1 4-1 --I +1 4-1 4-1 4-1 4-1 4-1 +1 +I 4-1 4-1 4-1 4-1

~ ea .4 o ~ ~ ~ ~ ~ ,-; o8 ,-.Z ~ ,--; ,,,4 ~ ~ ,-; ~ ~ ~ ,.4 ~ ~ ~

E
+l÷l+l+l+l+l+l+l+l+l+l+l+l+l+l+l÷l+l+l÷l+l÷l+l+l÷l +1 +1 ÷1 +1 +1 ÷1 +1 +1 +1

¢.q
I
o~
O

z
"O

<

+1 +1 ÷1 +1 ÷1 ÷1 +1 ÷1 +1 ÷1 ÷1 +1 +1 ÷1 +1 +1 +1 +1 +1 +1 +1 +t +1 +1 +1 4-14-14-14-1 -I-I +14-1 +i 4-1 O

t'q •,,, t"q .~ oq ~ ,--Z ~ eq

e~

[.-,
 BUILDING A TESTIS 141

+1+14-1+14-14-1+14-1+1+1+1+14-1+1+14-1+14-1

4-14-1 4-1+14-1+14-14-1+14-1+1+14-14-]+14-14-14-1+14-1+1+14-14-14-14-14-14-1÷1+1

o
4-14-1+14-1+14-1+14-1+1+14-1+14-1÷1 4-14-[ 4-14-1 4-14-1+14-1+14-1

4-1+14-14-1+14-1+14-1+14-14-14-14-1+1+14-1+14-14-14-14-14-1+14-1+1+1+14-1

+~ +14-14-14-14-14-14-14-14-14-14-1+14-14-14-14-1+14-1+1+14-1+[4-1+14-14-1 4-14-14-14-1

n O . ~ . . . . . . .
O
4-•4-•4-•4-•+•+•4-•4-I4-•+•+•4-•+•4-l4-•+•4-•4-•+•4-•+I••4-l+•4-•+•4-•••4-I4-•4-•+•

4-1 4-1 4-1 +1 4-1 4-1 4-1 +1 4-1 4-t 4-1 +1 4-1 4-1 +1 4-1
~2

+•4-•4-•4-•4-•+•4-•+•+•4-•+•4-•4-•+•4-•+•+l4-•4-•4-•+•4-•4-•4-l4-l4-•4-•4-•+•4-•4-•4-•
O
m~

4-1 4-1 4-1 4-1 +1 +1 4-1 4-1 4-1 +1 +1 4-1 4-1 4-1 4-1 4-1 4-1 +1 +1 +1 4-1 +! 4-1 4-1 4-1 4-1 +1 4-1

O
;>

+1 o
4-1 +1 4-1 +l +1 4-1 +1 +1 +1 +1 4-1 +i 4-1 4-1 +1 +1 +1 +1 4-1 4-1 4-1 4-1 +1 ÷1 +I +l +1 +1

o ~ ~ . ~ . ~ __~.o~.~.~.~..~.~.~.~..~.~.~.~. ~.
+1 +1 +1 q-I +1 4-1 +1 +1 4-1 4-1 4-1 4-1 +1 4-1 +l +l 4-1 +1-4-1 +1 +1 +1 +i 4-1 +l 4-1 4-1 4-1 +1 4-1
"5

©
;>
+l +l 4-1 +1 4-1 +i +l +l 4-1 4-1 +l +l 4-1 4-1 4-1 +1 +1 4-1 +l +l 4-1 +1 4-1 4-1 +1 +1 +l 4-1 4-1 +1 +1 4-1

~'2 O9
142 RUSSELL A N D DE FRAN(;A

0 ~ o
+13i+i~l

+l +l
,-*¢~

~ ~.~ +1+131+131+1
•~ ~

,= ~ . o +l+l+l+t+131

31~13131+13131+131÷1+131+1+1
'~ o,-~

3131+13131+1+131+1+1+1÷1

÷1+1+1+1+1+131+/+1+13131+1+1+1÷1

~ 1+1 +I +1 31 +1 +1 3i 31 +1 31 31 31+1+1÷1+1+1÷1+1

,x~ O
+131÷13131+1÷1+131÷1+i31+1+1÷1÷1+[+1

÷1 +i +t +1 +1 +1 31 +L +1 +1

/ +l+l+l+l+l+l÷l+131÷l+l+l÷l~l+l+l
O'~:3
~2

"d'~ +1 +t +1 +1 +1 31 +l +1 31 +1 31 31 +1 +1 +1 +1 ÷1 +1 +1 +1

rd ¢4

r~ r~
 BUILDING A TESTIS 143

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o

:~=o °

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oo
r~

o +l+l+l+l+l+l+l+l+l+l+l~l+l+l÷l~l+l +1 ÷~ ~1 ÷~ +l ÷l +I +1 ÷1 ÷1 ÷1 ÷1 ÷1 +1 ÷~ ÷1 +~
o

.9

+~
c~

. o ~.. ,= ~o
-H +l q-I -t-I q-I -H -l-I q-I -+-I q-I --t-I-H -+q q-I -+q -I-I -t-I q-I -H -t-I 4-1 +1 +1 -l-I -H q-I -H -H -I-I q-I q"l qq 4-1 +1

" ~ 5e~ o ~ o ~ r " ~ ° ,_~

° , - - ; ~ ,~~- - : r " ~,.~° ° , ~ , ~ , . _ ~ , _ , • e.i ~ e.i ~
05

o~
+l +1 +1 +1 +1 +l +1 ÷1 +1 +1 +l +1 +1 +1 ÷1 +I +l +1 +1 +1 +1 +1 +1 -H "t-I -H q-I +l -I-I -H q-I q-I q-I -H
• , ~ . .'~- .t ~ , o
~ ,z'~ ~ , ~::~ o e,~ ~ t ~ ~ , e~ ~ ; ,'~
o~
o

+1
+l +l +1 -I-I 4-1 ÷1 ÷1 +I -t-I +1 -t-I "~
o
g

c~
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r~

o
o

o
-I-I q'-I -t-I -I-I qq -t-I q-I -/-I "t-I +[ q-I

+l+l+l+l+l+I+l+l+l+l+l+l+l+l+l+l+l+l+l+l+l+l+l+l+l+l+l+l+l+l+l+l÷l+l
0

eq

r~

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r,.)
144 RUSSELL A N D DE FRAN(~A

+14-14-14-1 +1 +1 4-1 4-14-14-14-14-14-14-14-14-14-14-14-14-14-14-14-14-14-14-15-14-1

~ o~ ~ ~Z
. ~ ~Z_ ~ ~ . ~ ~Z ~o ~o ~ ~

.+ .~ ~
4-14-14-14-14-14-1 +14-1 +14-1 4-14-14-14-14-14-14-14-14-14-14-14-14-14-14-14-1 4-14-14-14-1

"a
~ O OO O ,-~ O tt~ ~ t",l ~ ¢'q O ,-,,~ O ~ O ~ O ~ O ~"~ O ¢¢~ O ¢'q O t"q O Cxl O t"xl O
4-14-14-14-14-14-14-14-14-1 +14-14-14-14-14-1 4-14-I +14-1 4-1 +14-1 +14-14-14-14-14-14-14-14-14-1
~.~ ~

. ,,...

+•+•4-i4-•4-•4-•+•4-•4-•4-•4-•4-•4-•4-•4-•4-•4-•4-•+•4-•4-•+•4-•+•+•4-•+•+•4-•+•4-•+•

o~

4-1 4-1 +14-1 4-14-14-14-14-14-1 +14-14-14-14-14-1

4-1 +14-14-14-14-14-14-14-14-14-14-14-14-14-1 4-14-14-t4-14-14-1+1+1 + 1 + 1 + 1 + 1 + 1 + 1 + 1 ÷ 1 + 1

O '~ +1 +1 +1 +1 +1 +1 +14-14-14-1 4-14-1 +1 + 1 ÷ 1 + 1 + 1 ÷ 1 + 1 + 1 ÷ 1 + l + l + l + l + l + l + t

5-1

+l+l+l+l+l+l+l+l÷l+l+l+l+l+l+l+l+l+l+l+l+l+l+l +1+1+1+1+1+1+1

Z~
&
o

+1+1+1+1+1+1+1+1+1+1+1+1+1+1+1 +1+1+1+1+1÷1+1+1÷1+1+1+1+1+1+1+I+1

c.~

~eq ~ ;~ ,,~ ~- r--- ~ "" ~ = & ~ '~ "~

 BUILDING A TESTIS 145

q-I q-I

+1+1+14-14-1--I

+1-t-I+1+1+1+1

+1 --I q-I +i ~ ~ q-I -H -H +1 +1 +1

+1 +1 q-I +1 +1 -t-I -H -H -H +1 +l -H

~.~ ~.~,~.~" ~.~ ~o~- ~'.o °G"

~ . o G'
° r~". ~ . ~
H -H +1 +[ +1 -H -H +1 +1 ~ -H +1 -H -H -H --I q-I q-I

~-}-I°4-14-1
~ o t--q_I _H¢5q-I o+~c'i~ °q-I~q
+1 ~4-1 ~[ ~ ~1 ~1 ~1 ~1 ~1 ~1

+l q-I -H +1 ~ ~ +1 -H +1 ~ ~ -H +1 -H -H +1 q-[ +1 -H +1

--I+1 q-I q-I -t-I q-I q-I ~

-E
!!
 146 RUSSELLAND DE FRAN(~A
to another. There are variety o f reasons for the varia-
bility seen, but we suspect that m a j o r variability has
only a few causes. First, the tissues utilized are prepared
by methods that result in tissue shrinkage. This
shrinkage m a y be differential. For example, Mori and
Christensen (1980), using glycol methacrylate as an
embedding medium, report the intertubular space to be
a b o u t twice the volume as most other reports (see
Table 4) that use epoxy resins as embedding media.
Secondly, the m o r p h o m e t r i c techniques utilized by inves-
tigators vary considerably. They are labor-intensive and
require n u m e r o u s calculations employing formulae in
which error is inherent. Thirdly, the a m o u n t o f sampling
used by individual investigators varies considerably.
Fourthly, interpretation o f micrographs whose quality
varies inevitably leads to differences in results. For
example, the difference between s m o o t h and r o u g h
endoplasmic reticulum is often a difficult determination
to make in some species, especially if tissues are p o o r l y
fixed. Categories where the data are reasonably similar
(e.g. see Leydig cell volume data in Table 5) for a specific
parameter lend m o r e confidence to the accuracy o f
the data.
For some, it m a y not be readily apparent h o w data
o f the kind provided in the Results and Discussion o f
this report have values o f physiological significance.
Firstly, these data are the ultimate structural descriptive
data o f the organ o f interest. The initial reports o f testis
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Franga, L.R., Hess, R., Cooke, P. and Russell, L.D. 1995a.
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structure were subjective. M o r p h o m e t r i c data provided
an objective assessment o f testis structure. Secondly,
m o r p h o m e t r y has only been relatively recently applied
to the testis; there have been interesting physiological
correlations of testis structure and function ( M o r i and
Christensen, 1980; Ewing et al., 1979; G h o s h et al.,
1992a, b; K u r o h m a r u et al., 1990; M e n d i s - H a n d a g a m a
et al., 1988, 1990a, b; Russell et al., 1994; Sinha Hikim
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b, 1993). Thirdly, biochemistry; and molecular biology
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testis. For example, the efficiency o f techniques which
allow isolation o f testis components, can only be judged
by knowledge o f the composition o f the intact testis.
The density o f enzymes and receptors can be determined
utilizing m o r p h o m e t r i c data. Fourthly, experimental
protocols that alter structural parameters can be under-
taken with data that are amenable to statistical analysis.
Thus, the data presented herein will serve as a ready
source o f information to fill a variety o f needs relating
to physiology, toxicology and pathology.
ACKNOWLEDGEMENTS
We thank the n u m e r o u s individuals cited f r o m our
laboratory that have, over the years, contributing data
to this paper and m a d e possible the building o f the
testis. Jim Stephens help in analysing the testis capsule
was especially appreciated.
in rats treated with propylthiouracil: Evidence that the Sertoli
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