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Morphometry of rat germ cells during spermatogenesis.

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THE ANATOMICAL RECORD 241:181-204 (1995)
Morphometry of Rat Germ Cells During Spermatogenesis
LUIZ RENATO DE FRANCA, SHI-JUN YE, LI YING, MEGAN SANDBERG, AND
LONNIE D. RUSSELL
Laboratory of Structural Biology, Department of Physiology, Southern Illinois University,
School of Medicine, Carbondale, Illinois (S.-J.Y., L.Y., M.S., L.D.R.); Department of
Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo
Horizonte, Minas Gerais, Brazil 31270-901 CP 2486 (L.R.DF.)
ABSTRACT
Background: There has never been a study of the components of germ cells as they progress through spermatogenesis.
Methods: The structural changes taking place in rat germ cells, from
spermatogonia to late spermatids, were studied utilizing morphometric
techniques conducted largely at the ultrastructural level.
Results: Volume and surface area parameters for virtually all cellular
and subcellular features were obtained for nine periods during the spermatogenic cycle. Virtuany an germ cen components show dynamic properties associated with specific phases of their development.
Conclusions: The data provided can be used in an objective way to characterize structural changes taking place during spermatogenesis and to
relate those structural changes to functional properties of germ cells.
0 1995 Wiley-Liss, Inc.
Key words: Testis, Morphometry, Germ cells, Spermatogenesis, Rat
Spermatogenesis is a complex process lasting about
two months in the rat. Cell divisions are characteristic
of the spermatogonial population of cells, insuring that
adequate numbers of sperm are available for male reproductive function. The unique feature of the spermatocyte population is the genetic recombination that
takes place, providing genetic diversity of the male gamete. Meiosis occurs in the spermatocyte generation of
cells to form a haploid male cell product that produces
a diploid cell upon union with the haploid female gamete. During the process of spermiogenesis, a dramatic
evolution of the spermatid occurs which takes the cell
from one which appears morphologically typical of a
relatively undifferentiated cell to a highly specialized,
elongate sperm possessing a flagellum, a n acrosome,
condensed chromatin and a highly elongate shape. The
uniqueness of this system makes it a n interesting
model for studies of organelle function and organelle
evolution with time.
The structural composition of germ cells should reflect the synthetic and degradative changes in cells as
they progress through spermatogenesis. Certainly, somatic cells in the testis show characteristic structure
that can relate to their function (Zirkin et al., 1980;
Mori and Christensen, 1980; Sinha Hikim et al.,
1989a,b; Kurohmaru et al., 1990). But there has never
been a quantitative analysis of the structural properties of the germ cell population in the testis of any
species as cells develop from spermatogonia to sperm.
One reason is the sampling procedures necessary to
perform morphometric studies which are labor intensive and require that large numbers of micrographs be
taken a t various stages of the spermatogenic cycle and
that a random sampling method be employed.
Q
1995 WILEY-LISS. INC
In the present report we use a proportional sampling
technique (Bugge and Ploen, 1986; Sinha Hikim et al.,
1989b) and montages constructed from nine periods
representing the fourteen stages of the cycle (Leblond
and Clermont, 1952; Russell et al., 1990) of the seminiferous epithelium to evaluate the cellular and subcellular components of germ cells during spermatogenesis in the rat.
METHODS
Animals
Four adult male Sprague-Dawley rats, weighing between 275 and 300 g (obtained from Charles River Laboratories, Wilmington, MA) were maintained in a photoperiodically regulated and temperature controlled
animal facility and given food and water ad libitum.
Tissue Preparations
Animals were perfused according to Sprando (Sprando, 1990). Under sodium pentobarbital anesthesia, the
left testis of each r a t was removed, weighed, and its
volume measured by water displacement (Steer, 1981).
This testis was fixed with 5% glutaraldehyde in 0.05 M
cacodylate buffer (pH 7.4) preceded by a brief saline
wash. After glutaraldehyde fixation, the right testis
was diced into small pieces, placed into the same fixa-
Received May 23, 1994; accepted August 2, 1994.
Address reprint requests to Dr. Lonnie D. Russell, Laboratory of
Structural Biology, Department of Physiology, Southern Illinois University, School of Medicine, Carbondale, IL 62901-6512.
This work was supported in part by a fellowship awarded to Luiz
Renato de Franqa from the Brazilian Research Council (CNPq).
182
L.R.
IE
FRANCA ET AL
TABLE 1. Volumes (pm3)of individual germ cells (mean & SE)'
Stages
I
11-IV
V
VI
VII
VIII
IX-XI
XII-XI11
XIV
Suermatoeoniaiurimarv
spermatocytes
492 t 30 (type A I-XIV)
433 t 40 (In I-IV)
352 t 23 (type B V-VI)
(see stage V)
332 t 37 (PI)
468 t 50 (PI)
574 t 36 (L)
497 t 40 (Z)
766 t 59 (PI
PrimarviSecondarv
spermatocytes
658 t 135 (P)
1,112 t 149 (P)
1,541 2 134 (P)
1,778 2 172 (P)
2,498 t 118 (P)
2,816 2 223 (P)
2,624 2 225 (P)
4,202 t 123 (PiDi)
2,507 t 220 ( S )
Roundielongate spermatids
1,279 2 86 (step 1)
1,443 t 38 (step 2-4)
1,690 i 169 (step 5)
1,901 i 142 (step 6)
1,914 ? 73 (step 7)
1,513t 116 (step 8 )
1,277 t 31 (step 9-11)
1,164 t 138 (step 12-13)
1,189 97 (step 14)
Elongate spermatids
1,198 t 100 (step 15)
1,262 t 80 (step 16-17)
1,105 t 172 (step 17)
899 k 81 (step 18)
580 t 115 (step 19)
452 t 106 (step 19)
'Volumes of germ cells listed by type including flagella. Data from germ cell volumes from stages XII-XI11 (zygotene) through step 19
spermatids were previously reported (Franqa et al., 1993)without the addition of their flagellar volumes to the cell volumes. Abbreviations used
in the tables to refer to germ cell types are provided in the legend to Figures 1-10,
tive for a n additional hour, washed in cacodylate buffer
overnight, post-fixed with 1% osmium:l.25% potassium ferrocyanide mixture (Russell and Burguet,
1977), dehydrated in ethanol, and embedded in Araldite (CY 212). During embedding, tissue blocks were
oriented such that seminiferous tubules could be sectioned transversely.
For electron microscopic studies, thin sections showing a silver to pale gold interference color were cut on
a Reichert ultramicrotome (Ultracut-E, Reichert-Jung,
Vienna, Austria) with a diamond knife, mounted on
Formvar-coated slot grids, and stained with uranyl acetate and lead citrate.
Morphometry
Montages were reconstructed from micrographs, the
negatives of which were taken at high ( x 9,000) magnification. The final magnification of micrographs was
x 22,140. Thirty-six high magnification montages
were constructed of portions of seminiferous tubules a t
nine time periods. Thus a total of approximately 3,200
micrographs were utilized in the nine time periods selected, represented by stages I, 11-IV, V, VI, VII, VIII,
IX-XI, XII-XIII, and XIV, during the cycle of the rat
seminiferous epithelium (Leblond and Clermont, 1952;
Russell et al., 1990). Proportional sampling employing
pie-shaped wedges of the epithelium was undertaken
(Bugge and Ploen, 1986; Ye et al., 1993). The position
of the pie-shaped wedge in the seminiferous tubule was
selected according a lottery method (Sinha Hikim et
al., 1989b). High magnification montages encompassed
about one eighth of the tubule. In addition, wedgeshaped montages were taken at the lumen in stage VIII
for germ cells released from their residual cytoplasm a s
sperm.
Most germ cell volumes utilized in the present study
were those previously determined by FranGa et al.
(1993). The volumes of some germ cells (spermatogonia, preleptotene, and leptotene spermatocytes) were
determined in the present study by first serially reconstructing nuclei a t the light microscope level as has
been previously described (Sinha Hikim et al., 1988),
determining the volume of the nuclei by summing the
areas obtained of all sectioned profiles and finally
at the electron microscope level by obtaining a ratio
of points of the cytoplasm to the nucleus of the cell.
The product of the nuclear volume and the aforemen-
tioned cytoplasminucleus points ratio yielded the cell
volume.
Some germ cell types were poorly represented in tissue section used to construct montages because of their
infrequent occurrence. In some instances it was necessary to photograph images of Type A, intermediate,
Type B spermatogonia, preleptotene, leptotene, and
pachytene spermatocytes in areas outside the montage
to obtain sufficient micrographs of these cells for a determination of cytoplasminucleus ratio and for determination of parameters relating to organelles. In those
instances, all images of these cell types were photographed and examined in a predetermined length
along the base of the tubule. Type A spermatogonia
were photographed at random from the various tubules. No attention to specific subtype of Type A spermatogonia was made since morphological criteria are
not available to differentiate the various subtypes of
these cells in sectioned profiles. Thus the data obtained
represent a mean for Type A spermatogonia a t all
stages of the cycle. The mean is considered a composite
of the spermatogonial cell types that has a relationship
to their frequency of occurrence since spermatogonia
were photographed at all stages of the cycle.
The volume densities and surface densities of selected organelles was determined in high magnification montages by point and intersection counting
methodology using a multipurpose grid (line lengths of
1 cm and 1.5 cm). The volume density percent was expressed as the number of points over a particular feature divided by the total points over the cell (nucleus
plus cytoplasm) containing that feature times 100. The
volume of a n structural component was determined as
the product of the volume density and the cell volume.
The formula used to determine surface density is as
follows:
4 x 1
s, = P, x 2'
where I is the number of intersections on the membrane and P, is the number of points over the germ cell
and Z is the length of the line between points in terms
of magnification of the micrograph.
To determine surface (S) area from surface density,
the following formula was applied:
s = s,
x
v,
where V equals the volume of the germ cell
183
GERMCELLMORPHOMETRY
TABLE 2. Spermatogonia and spermatocytes volume parameters (mean f SE); volume (pm3)and volume density
in Darentheses
~
Germ
cell
type
or
stage
A
I-XIV
In
I-IV
B
v-VI
PI
VII
PI
VIII
L
IX-XI
Z
XII-XI11
P
XIV
P
I
P
11-IV
P
V
P
VI
P
VII
P
VIII
P
IX-XI
PiDi
XII-XI11
S
XIV
~~
Golgi
Ground
substance
Nucleus
Smooth
endoplasmic
reticulum
183 t 14
(37.1 t 1.3)
181 f 38
(40.8 f 4.6)
129 t 18
(36.5 f 3.3)
127 t 29
(37.0 t 4.0)
155 f 27
(32 8 t 3.9)
175 f 23
(30.2 t 2.1)
166 f 17
(33.3 f 1.4)
268 f 12
(35.8 f 3.4)
211 f 55
(30.5 f 3.2)
474 f 82
(42.2 f 4.8)
734 f 81
(47.2 f 1.5)
835 f 74
(47.2 f 2.4)
1182 f 74
(47.2 f 1.7)
1434 f 131
(51.2 f 4.4)
1295 2 175
(48.8 t 2.7)
2345 f 126
(56.1 f 3.7)
1681 f 103
(68.2 f 6.0)
269 t 13
(55.0 f 1.3)
223 f 8
(52.5 t 4.8)
192 f 17
(54.7 t 4.5)
159 f 3
(49.2 f 4.1)
240 f 20
(52.2 f 5.5)
322 f 10
(56.3 f 1.9)
253 f 18
(51.0 f 1 2 )
402 f 59
(51.6 t 4.3)
350 t 63
(56.0 2 5.8)
446 t 76
(40.9 t 7.2)
506 2 61
(33.0 t 2.7)
679 t 120
(37.3 t 4.6)
736 t 41
(29.4 t 0.7)
799 t 144
(28.1 t 4.2)
891 t 61
(34.4 f 2.6)
1141 t 215
(27.0 t 4.7)
492 t 226
(18.0 2 7.0)
15 f 2
(3.0 f 0.2)
15.6 f 3
13.6 f 0.4)
12.2 f 2.3
(3.5 f 0.7)
25 f 6
17.4 f 1 6 )
32 f 7
(6.8 2 1.4)
38 f 5
(6.5 2 0.6)
47 f 4
(9.6 f 0.7)
43 t 7
(5.7 t 0.8)
47 2 14
(6.9 2 1.2)
104 t 22
(9.2 t 1.5)
119 t 14
17.9 t 1 0 )
106 f 10
(6.1 f 0.6)
282 t 29
(11.3 t 0 9 )
303 f 41
(10.7 t 0.9)
200 f 27
17.6 t 0.8)
373 2 63
(8.9 5 1.5)
194 2 34
(8.1 t 1.9)
+
Rough
endoplasmic
reticulum
Mitochondria
associated
vesicles
4 5 2 0.7
(0 9 2 0.1)
3 3 t 0.8
(0 7 t 0.1)
3.9 t 1.2
(1.1 2 0.3)
3.0 f 1.3
(1.0 t 0.5)
8.2 2 3.6
11.6 t 0.6)
5.5 t 1.6
(1.0 t 0.3)
3.9 t 0.8
(0.8 2 0.1)
9.2 5 0.5
(1.2 f 0.1)
7.3 t 2.2
(1.1f 0.2)
15 t 3
(1.5 t 0.3)
20 f 4
(1.3 f 0.4)
21 t 4
(1.3 t 0.3)
29 r 7
(1.1 f 0.3)
19 f 3
(0.7 f 0.1)
22 t 3
(0.8 2 0.1)
43 t 12
(1.0 t 0.31
30 t 3
(1.25 f 0.2)
16 f 3
(3.2 f 0.5)
8 5 f 0.8
12.0 f 0.1)
11.3 f 1.9
13.2 f 0.4)
15.1 f 4
(4.4 f 0.6)
15.1 f 4
(3.1 2 0.6)
17 f 4
(2.9 2 0.6)
14 f 2
(2.8 2 0.3)
22 2 6
(2.8 2 0.7)
24 t 8
(3.2 t 0.8)
56 5 13
15.0 5 1-01
79 2 7
1 5 2 r 06)
83 t 10
( 4 9 t 1.0)
176 5 26
(7.1 f 1.1)
166 t 18
(5.9 2 0.4)
139 t 5
(5.4 f 0 4)
193 f 21
14.6 5 0.4)
93 2 6
(3.8 t 0.2)
1.6
(0.9 2 0.3)
1.6 f 0 5
(0.4 t 0.1)
3.6 2 0 4
(1.0 f 0.11
2.1 f 1 6
(0.8 r 0.6)
17 f 7
13.4 t 1.0)
14 t 9.6
12.6 t 1.7)
1125
(2.1 2 0.9)
20 t 7
(2.7 2 1.0)
14 t 9
(1.7 f 1.2)
14 f 4
10.9 f 0.9)
69 t 36
(Mf 2 1 )
36 f 7
(2.1 f 0 5)
79 f 12
(3.3 f 0.6)
67 f 20
(2.4 f 0.7)
58 f 10
(2 2 f 0.4)
82 f 30
I19 f 0.7)
14.1 f 5.1
(0.55f 0.19)
Approximately 2,000, 1,500, and 4,000 points were
used to sample spermatogonia, spermatocytes, and
spermatids in each cell type a t each stage for each animal, respectively. The total number of points recorded
for all germ cell types was approximately 320,000.
Not all organelles were readily recognizable. They
are described in the Results Section of this paper. For
volumetric determinations (and surface area determinations) these structureslorganelles were given names
and quantitative data recorded for them.
A shrinkage correction factor of 1.189 was applied to
volume data in the present study. This correction factor
was determined in a previous experiment using a similar fixation protocol (Sinha Hikim et al., 1988) and has
varied little in other subsequent experiments from this
laboratory.
A mean and standard error were calculated using for
animals for each parameter except where noted in the
tables.
RESULTS
Germ cell volumes are presented in Table 1. Data
from spermatogonia and some spermatocytes are combined with data from FranGa et al. (1993) to provide a
comprehensive picture of the cell volumes throughout
spermatogenesis from which the subsequent subcellular volume and surface area data are based. The data
on cell volumes in the present study, however, are
slightly different than those in the previous study
(FranGa et al., 1993) since flagellar volumes obtained
in the present study were added to them.
Table 2 provides the data on volume density and vol-
4.3
2
Lysosome
Discoid
Nuage
(all types)
Vesicles
associated
with
chromatoidlike body
Lipid
0 08 f 0.08
(0 02 t 0.021
0.8 f 0.4
(0.2 f 0.1)
0 7 f 0.4
(0.13 f 0 08)
2.0 f 1 2
(0.34 f 0.20)
0.3 f 0 3
(0.05 t 0.05)
0.5 f 0.3
(0.06 2 0.03)
1.1 2 0.8
(0.14 t 0.09)
3.3 t 0.6
@.?a 2 0 . w
2.4 f 0.8
(0.15 t 0.05)
4.1 t 1.1
(0.17 t 0.05)
6 2 2.0
(0.2 t 0.06)
1.7 t 1.0
(0.06 t 0.04)
3.5 2 0.9
(0.08 f 0.02)
4.4 t 0.8
(0.17 t 0.03)
0.2 2 0.2
06206
(0 03 2 0.03) (008 t 0 08)
0 9 2 0.5
0.9 t 0 3
(0.2 t 0.1)
(0.2 t 0 06)
0.2 t 0.2
07t07
(0.03 f 0.03) (0 08 f 0 08)
2.1 t 0.5
14209
(0.2 f 0.1)
10.3 2 0.04)
1.1f 0 7
1.9 t 1.1
(0.2 f 0.1)
(0.11 f 0.07)
7.0 f 1.2
3.9 f 0 4
@.a6
6.061 e.5
c 8.85)
4.9 f 0.5
11.1 t 0.9
(0.28 f 0 02)
(0.7 2 0.1)
14f08
7.4 f 2.7
(0.06 f 0.03) (0.30 f 0.12)
2 0 t 0.2
20 f 6.0
(0.07 f 0.04)
(0.7 f 0.1)
1.6 f 0.7
16 f 3.0
(0.06 f 0.03)
(0.7 f 0.1)
0.7 f 0.4
18 f 3.0
(0.02 f 0.01) (0 4 f 0.06)
2.5 t 1.4
10.1 f 0.06)
*
0.5 f 0.5
(0.02 t 0.02)
0.3 t 0.3
10.01 c 0.01)
2.2 2 1.4
10.05 t 0.031
1.0 2 0.4
(0.04 t 0.01)
1.4 2 0.9
(0.06 2 0.04)
0.5 2 0.5
(0.01 r 0.01)
ume of spermatogonial and spermatocyte subcellular
components expressed on a per cell basis. Table 3 shows
the comparable data on the same parameters in spermatids. Table 4 displays the data on surface density
and surface area for spermatogonia and spermatocytes
and Table 5 provides the comparable data on the same
parameters in spermatids.
In Figures 1 through 10 the data are plotted and
best-fit line graphs (fourth-order polynomial) of the
data are shown for ease of visualization of trends taking place during spermatogenesis. Figure 11 provides
volumetric data on sperm released from their residual
bodies, but not yet released from the Sertoli cell. This
figure also depicts the volumetric composition of residual bodies.
Cell
In the series of cell divisions that take place among
type B and intermediate spermatogonia to form preleptotene spermatocytes, there was a decrease in cell size
such that stage VII preleptotene spermatocytes were
approximately 77% of the volume of intermediate spermatogonia. From preleptotene, individual spermatocytes increased in size and a t stage V the rate of volume increase was accelerated through diplotene of
meiotic prophase (Fig. 1). Cell size was approximately
halved after the first, and also after the second meiotic
division. During early spermiogenesis the round spermatids increased in volume until step 7 where a sharp
decline was noted beginning at step 8. The cell volume
stabilized from stage IX-XI (steps 9-11) until stage V
(step 17) where a sharp decline in volume once again
184
L.R.
DE
FRANCA ET AL.
Table 3. Spermatid volume parameters (mean 2 SE);
Golgi
Step
Ground
substance
Or
stage
1
762 i 60
(59.7 f 3.31
854 t 10
11-IV
(59.3 t 1.31
5
966 t 95
V
157.2 t 0.5)
1159 t 39
6
VI
161.7 i 3.6)
7
1133 i 44
VII
159.3 i 2.1)
8
984 i 94
VIII
(65.0 t 2.81
9-11
883 f 32
IX-XI
(69.2 t 2.01
12-13
841 t 95
XII-XI11 (72.7 i 1.4)
14
903 i 87
XIV
175.8 i 1.5)
15
894 t 68
I
174.9 t 2.5)
16-17
986 t 72
11-IV
178.0 i 1.1)
17
856 i 138
V
(77.3 t 1.61
696 t 75
18
VI
177.5 t 1.8)
432 t 84
19
VII
(74.8 t 1.9)
19'
273 t 59
VIII
(61.0 t 4.01
Residual'
186 i 6
bodv VIII 162.5 t 0.71
I
2-4
'N
'N
=
=
Nucleus
Acrosome
356 2 40
127 8 2 1 9 )
375 2 19
126.0 2 111
439 2 46
126.1 2 1.81
440 2 91
122.6 2 3.5)
536 2 43
(27.9 2 1.61
268 2 53
(17.9 2 3.4)
123 2 42
(9.7 2 3 3)
31 2 10
(2.7 2 0.8)
14 t 4.6
11.2 z 0.5)
18.5 2 2.9
11.5 2 0 2)
19.3 2 9.3
11.6 2 0.81
11.4 2 2.4
(1.1 2 0.2)
17.7 2 7.1
12.0 2 0.8)
12.2 2 4.6
(1.9 2 0.6)
9.1 2 1.4
(2.1 2 0.31
5.8 2 3 8
(0.4 2 0.2)
10 2 3.0
I0 6 2 0 2)
14 2 4.8
(0.7 2 0.21
22.1 2 2.3
11.2 t 0.09)
15.8 t 2.5
11.1 2 0.21
16 5 2 3.8
(1.3 z 0.31
15.1 t 3.9
(1.4 t 0.4)
11.2 t 3.1
(1.0 t 0.3)
14.2 t 1.7
11.2 t 0.091
15.1 t 6.1
11.3 t 0.5)
7.1 t 1.9
(0.7 f 0.2)
11.1 f 4.1
11.3 t 0.5)
7.7 2 4.3
(1.1t 0.5)
6.4 t 1.5
11.4 f 0.31
Subacrosoma1
space
Endoplasmic
reticulum
+
Radial
body
89 t 18
( 6 9 2 1-31
124 2 11
03 2 02
10 02 2 0 011 18.5 t 0.51
170 2 33
1.0 2 0 6
10.07 2 0 051 I9 9 2 1.11
14203
179223
10.07 2 0.01) (9.3 t 0.6)
3.1 2 0.8
136 t 15
(0.17 t 0.05) (7.1 f 0.7)
142 t 15
2.4 t 1.4
2.4 t 0.6
10.16 2 0.03)
(9.3 t 0.5) (0.17 t 0.111
39 2 04
145 t 14
3.2 f 2.3
(0.31 t 0.031 (11.3 f 1.01 10.24 t 0.17)
131 i 22
4.8 2 2.2
5.0 t 1.5
10.47 t 0.171 (10.9 2 0.7) (0.36 ? 0.161
3.5 i 1.8
122 2 15
5.8 2 2.1
(0.31 ? 0.171 110.3 i 1.3) 10.49 t 0.171
3.6 t 1.1
118 i 25
3.8 2 1.6
(0.29 t 0.08) (9.7 t 1.6) 10.23 t 0.11)
118 i 14
2.5 t 1.8
3.8 t 1.2
(0.32 t 0.11) 19.3 i 0.61 (0.19 z 0.121
95 f 19
2.7 ? 0.3
2.2 t 0.7
(0.20 f 0.07) (8.5 t 0.61 10.26 t 0.05)
56 t 9
1.4 i 0.5
4.7 t 1.0
(0.55 t 0.13) (6.3 t 0.8) 10.17 t 0.08)
2.6 2 1.8
7t2
(0.38 t 0.23) (1.1 f 0.21
1.6 t 0.3
4.6 i 1.6
(0.32 f 0.021 10.9 f 0.1)
2.5 i 1.7
10.8 t 0.6)
Mitochondria
associated
vesicles
55 2 8
14 3 ? 0.51
70 2 9
14.8 2 0 51
78 2 9
I4 6 2 0 3)
88 2 10
14.7 f 0.4)
63 t 2
(3.3 t 0.11
77 t 11
15.0 t 0.51
69 t 5
15.4 t 0.41
75 i 11
(6.4 ? 0.41
67 2 8
(5.6 2 0.4)
76 ? 6
16.4 i 0.5)
61 i 6
14.9 i 0.5)
68 t 15
(6.1 I
0.61
50 t 10
15.5 t 0.81
28 t 8
14.7 t 0.8)
34 t 9
(7.4 t 0.71
10.7 t 0.7
13.6 t 0.07)
7.6 i 1.6
10.58 t 0.09)
5.9 f 2.1
(0.40 t 0.131
11.7 t 3.5
10.72 t 0.251
8.5 i 2.2
10.44 i 0.11)
10.0 i 2.6
10.51 t 0.12)
10.6 t 1.1
(0.71 ? 0.071
9.7 i 1.3
(0.75 2 0 091
9.2 2 2.8
10.75 2 0 16)
9 6 2 1.4
10.83 2 0.161
90 2 16
10.74 2 0 09)
58 2 28
(0.44 2 0.211
3.8 2 0.6
10.37 t 0.071
0.4 t 0 3
10.04 C 0 03)
Lysosome
Multivesicular
body
Lipid
1.0 ? 0.3
0.2 2 0.1
0.08 2 0 08
10.08 i 0.031
(0.02 2 0.01) (0.005 2 0.0051
1.4 ? 0.4
0.1 2 0.1
(0.1 ? 0.031 (0.008 2 0.0081
3.0 ? 1.4
0.2 2 0.2
1.3 2 0.9
10.16 i 0.06)
10.02 2 0 021
(0.07 2 0.05)
1.7 t 0.7
0.7 2 0.4
1.4 2 1 0
(0.09 2 0.03)
10.04 2 0.03)
I0 08 2 0 06)
0.7 2 0.1
0.5 2 0.3
54218
10.04 2 0 0081 I0 03 2 0.02)
(0.28 2 0.091
11 2 0.1
0 4 2 0.2
4721.2
10.07 2 0.011
(0.03 2 0.02)
(0.31 2 0 08)
0.8 2 0.3
0.3 2 0.2
61208
(0.03 2 0.011
(0 06 2 0 03)
(0.48 2 0 06)
1.3 2 0.7
5.5 2 1.1
I0 13 2 0.07)
10.47 2 0.07)
2 5 2 0.5
0.2 i 0.1
5.4 t 0.3
10.21 2 0.041
10.02 i 0.01)
10.47 t 0.06)
0.7 2 0.5
8.5 2 2.1
(0.07 2 0 061
10.68 2 0.131
2.7 2 0.7
0.8 t 0.3
20.0 2 2.4
I0 22 2 0.06)
10.06 t 0.02)
(1.58 2 0.151
1.0 2 0 5
25.2 2 5.5
I0 08 2 0.031
I2 4 2 0 6)
0.4 2 0.4
26.3 2 9.5
10.05 2 0 051
12.8 t 1.11
0.2 2 0.2
4.4 2 1.8
17.8 2 3.7
(0.03 2 0 031
(1.1t 0.5)
13.0 2 0.21
1 6 2 0.9
24.0 2 5.4
2.9 2 1.3
(0.57 2 0 131
I0 46 2 0.231
15.5 2 0 6)
27209
4.0 2 4.0
19 0 2 1 0
10.9 t 0 31
11.3 2 1.31
(6 4 2 n fii
Chromatoid
body
5.5 t 1.9
(0.42 t 0.14)
2.2 t 0.6
(0.16 t 0.041
5.5 2 1.1
(0.33 2 0.061
5.2 t 1.1
10.28 t 0.06)
2.3 f 0.9
10.12 t 0.051
2.6 t 0.9
10.17 t 0.06)
1.3 t 0.09
10.10 i 0.05)
0.5 t 0.3
(0.04 2 0.02)
0.2 t 0 2
(0.02 2 0.02)
3.
2.
occurred through step 19 spermatids. The volume of
the step 19 spermatid (containing residual cytoplasm)
was approximately one-third of that of a step 1 spermatid.
Throughout spermatogenesis, the surface area of the
cell (plasma membrane) generally paralleled the increase in volume of cells (Fig. 1).During the elongation
phase of spermiogenesis, as cell volume decreased, the
cell surface area markedly increased. For example, a
step 15 spermatid showed approximately twice the surface area as did a step 1 spermatid. Surface area remained constant until step 18 (in stage VII) when a
22% reduction in surface area ocurred from step 18
spermatid to a step 19 spermatid (of stage VIII).
Nucleus
In the rapid spermatogonial divisions that precede
the formations of preleptotene spermatocytes, the nuclear size remains relatively stable (Fig. 2). A trend
toward nuclear growth is evident in preleptotene cells
in stage VIII as compared with the same cells in Stage
VII. Subsequently, nuclear growth is progressive and
continues throughout spermatocyte maturation until
diplotene. Nuclear volume in secondary spermatocytes
is less than half of that in its diplotene predecessor.
Mean nuclear volume of a step 1 spermatid is 72% of
that of secondary spermatocytes.
Spermatid nuclei increase in volume by about 50%
from step 1 to step 7 of spermiogenesis. At step 8, at
steps 9-11, at steps 12-13, and step 14 there are
marked decreases in nuclear volume as compared with
the previous stage (50%, 54%, 75%, 55%, respectively).
The nuclear volume remained relatively stable until
step 19. The nuclear volume of sperm was determined
to be 9.1 pm3.
Surface area changes in the nuclear membrane paralleled nuclear volume changes until early pachytene
(about stages 11-IV) where the nuclear envelope increased its surface area in relative comparison with the
nuclear volume (Fig. 2).
At step 8 of spermiogenesis, a reduction in nuclear
surface area occurred that continued to step 14 such
that the step 14 spermatid displayed only approximately 30% of the step 1 nuclear surface area.
From step 14, nuclear surface area remained stable
until step 19 where a n approximate 47% decrease occurred. There were generally similar trends in nuclear
surface area and volume throughout spermatogenesis.
It was noted at late step 7 and step 8 of development
that there was a n inpocketing of the nucleus of spermatids (Fig. 12). The involuted nuclear membrane appeared convoluted and without normal cytoplasmic
constituents. Assuming the invaginations were part of
the nucleus (which they are not), the volume of the
nucleus found to be taken up by such cytoplasmic inpocketings was about 1-0.5% in step 7 and step 8, respectively. Nuclear inpocketings were not seen in step
9 spermatids.
Ground Substance
During early spermatogenesis, the volume of the organelle-free portion of the cytoplasm paralleled the
changes in the volume of the nucleus (Fig. 2). The trend
however differed during the pachytene phase of sper-
185
GERM CELL MORPHOMETRY
volume (wm3)and volume density in parentheses
~~~~
Vesicles
associated
with chromatoid body
Granulated
body
Reticulated
body
Dense
fibers and
fibrous
rine
Myelin
fieure
Clear vesicles
associated
with endoplasmic
reticulum
Clear
associated
vesicles
Vesicles
associated
with
dense
material
Lightly
granular
vesicle
Vesicles
with
medium
density
Tubulobulbar
complex
Endop1asm ic
reticulum
close to
manchette
2 0.8
(0.21 2 0 061
2.7
2.4 2 0.4
(0 17 2 0.021
2.6 2 1.0
(0.14 2 0.041
1.1 2 0.5
(0.07 2 0.03)
0.8 2 0.4
10.04 2 0.021
1.0 2 0.3
(0 06 t 0.02)
2.8 t 0.9
(0.22 t 0.071
4.0 t 0.7
(0.34 ? 0.04)
1.5 2 0.7
(0 13 2 0.06)
0.9 t 0.4
(0.08 t 0.04)
0.6 2 0.2
(0.06 2 0.031
1.8 2 0.7
1.7 2 1.0
10.15 2 0.05) (0.13 2 0 06)
2 2 2 0.7
2.9 f 1.1
I0 19 2 0.06) (0.26 f 0.12)
2.1 2 0.4
0.8 t 0.2
(0.17 2 0.041 (0.06 t 0.01)
2.6 2 1.0
0.7
-t
0.3
(0.08 t 0.031
(0.24 2 0.1)
0.6 t 0.2
2.4 2 0.9
(0.29 C 0.12) (0.06 2 0.02)
0.4 t 0.3
(0.07 2 0 041
0.2 2 0.07
(0.04 2 0.02)
3.6 t 0.7
(0.28 t 0.05)
4.8 t 1.8
(0.41 t 0.131
3.8 2 0.6
(0.32 2 0.051
13.2 2 5.2
(1.04 t 0.391
14.0 2 1.0
(1.12 2 0.081
19.1 2 4.1
11.8 2 0.451
19.3 2 3.6
(2.14 2 0.31
336~113
(6.0 Z 151
58.4 2 22.0
(11.7 Z 2.21
2.2 2 0.7
(0.15 f 0.051
1.5 t 1 1
10.08 t 0.051
1.1 t 0.5
(0.06 ? 0.03)
0.7 ? 0.2
(0.04 t 0.009)
0.6 2 0.6
I0 04 t 0.04)
1.2 2 0.8
(0.09 z 0.06)
0.8 2
10.06 2
0 12
(0.01 2
02 Z
(0.01 2
0.4
0.03)
01
0 01)
0.1
0.0081
0.2 2 0.2
10.02
2
0 021
7 3 2 4.8
(0.55 2 0.35)
29.9 2 15.7
(2.5 2 131
26 9 2 2.0
( 2 3 2 0.28)
28.3 2 7.1
12.3 f 0.42)
3.1 f 1.3
(0.25 C 0.111
2.7 2 1.8
(0.28 f 0.201
9.9 f 3.4
(1.06 2 0.281
8.5 t 3.2
(1.27 2 0.351
matogenesis where ground substance grew in volume
much more rapidly than did the nucleus.
During spermiogenesis, whereas the nucleus markedly declined in volume in steps 8-14 and remained at
a low volume, there was only a gradual reduction in the
volume of ground substance at step 8; a rather constant
volume from step 8 to step 17 (stages 11-IV); and a
marked reduction in the ground substance from step 18
(stage VI) to step 19 (stage VIII). The volume of the
ground substance at step 19 (at stage VIII), expressed
on a per cell basis, was approximately 36% of that of
step 1 spermatids.
The volume of the ground substance of the residual
body, a s determined from two of the four animals studied, comprised 68% of the volume of the ground substance of step 19 spermatid (in stage VIII). The volume
of the ground substance of spermatids lacking connection to residual bodies was 57.8 ~m~ (Fig. 11).
Smooth Endoplasmic Reticulum
The surface area of smooth endoplasmic reticulum
(Fig. 31, a s expressed per cell, increased substantially
(approximately 25 times) in germ cell development
from type A spermatogonia to diplotene spermatocytes.
The surface area of smooth endoplasmic reticulum approximately halved during the two meiotic divisions.
During spermiogenesis the surface area of endoplasmic
reticulum increased slightly in young spermatids until
step 6 of spermiogenesis and declined slowly thereafter
until steps 16-17 (in stages 11-IV) where a n accelerated decrease occurred in subsequent steps. From its
greatest surface area in pachytene a n overall 52-fold
depletion of this organelle by step 19 (in stage VIII). (It
should be noted that smooth and rough endoplasmic
0.1 2 0 1
(0.008 2 0.008)
3.9 f 1.5
1.2 2 0.5
I0 31 t 0.11
(0.09 2 0.04)
5.9 2 0.6
1.7 2 0 6
(0.51 f 0.07)
10.14 2 0 041
3.3 ? 1.4
1.2 2 0.2
10.25 2 0.10)
(0.10 f 0.02)
4.4 2 1.6
2.1 2 0.6
(0.33 2 0.11)
(0.17 2 0.041
4.7 Z 1.5
2.2 t 0.9
(0.4 2 0.1)
(0.20 2 0.08)
1.8 2 0.8
0.6 2 0.3
(0.18 t 0.07)
10.07 t 0.041
3 2 2 10
2 0 12)
09 2 09
(0 06 2 0 061
I0 32
22.8 2
(4.14 2
35.1 2
(8.5 C
71.3 C
(24.0 t
8.7
1.3 Z 0.6
1.4 2 0.5
1.12) 10.24 2 0 091 (0.23 2 0.05)
6.5
0.6 2 0 3
1.81 (0.17 2 0.08)
0.6
1.5 2 0.3
0.8)
(0.5 t 0.1)
reticulum were considered together in spermatids since
it was difficult to distinguish varieties of the endoplasmic reticulum in these particular germ cells.)
In general, the volume of the smooth endoplasmic
reticulum paralleled that of its surface area throughout spermatogenesis.
Endoplasmic reticulum of the saccular variety was
seen adjacent to the manchette microtubules (Fig. 13A)
primarily in steps 12 and 13 and to a lesser extent in
step 14. This variety, although different in appearance
from that seen throughout most of the cytoplasm, was
combined into the general category of endoplasmic reticulum. In the subsequent stages up until step 15 of
spermiogenesis, large cisternae resembling those adjacent to the manchette were seen free in the cytoplasm
(Fig. 13B) or adjacent to the radial body (Fig. 16A).
In steps 16 through 18, membranous structures resembling endoplasmic reticulum were noted in which
individual saccules were small, with highly collapsed,
parallel membranes compared with other endoplasmic
reticulum in the cytoplasm (Fig. 14). These small, collapsed saccules were scattered throughout the cytoplasm. These structural elements were classified a s endoplasmic reticulum although there was the possibility
that the structural components seen were parts of a
dissociating Golgi apparatus (Oko et al., 1993).
Rough Endoplasmic Reticulum
Rough endoplasmic reticulum had a saccular form
early in spermatogenesis and was distinctly different
in appearance when compared with the smooth variety.
However, during spermiogenesis, it was difficult to distinguish the rough variety from the smooth endoplasmic reticulum, especially using a n osmium:ferrocya-
186
L.R.
I)E
FRANCA ET AL.
TABLE 4. Spermatogonia and spermatocytes surface parameters (mean 2 SE); surface area (prn') and surface
density in parentheses
Germ
cell
type
or
stage
A
I-XIV
In
I-IV
B
v-VI
PL
VII
PL
VIII
L
IX-XI
2
XII-XI11
P
XIV
P
I
P
11-IV
P
V
P
VI
P
VII
P
VIII
P
IX-XI
PiDi
XII-XI11
S
XIV
Golgi
Plasma
membrane
Nuclear
membrane
Smooth
endoplasmic
reticulum
308 t 17
(0.63 t 0.031
259 t 47
(0.59 t 0.051
276 t 26
(0.78 t 0.051
201 t 28
(0.60 t 0.031
259 t 54
(0.54 t 0.091
419 t 65
(0.74 t 0.11)
273 t 30
(0.55 t 0.03)
477 2 18
(0.63 t 0.041
386 t 81
(0.58 t 0.021
716 t 237
(0.60 t 0.12)
895 t 119
(0.58 t 0.041
858 t 41
(0.49 t 0.02)
1110 t 106
(0.44 t 0.03)
1062 t 30
(0.38 t 0.02)
940 t 41
(0.36 2 0.031
1393 t 147
(0.33 t 0.03)
1358 t 138
(0.55 2 0.061
199 t 8
(0.41 t 0.02)
163 t 21
(0.37 t 0.021
158 t 19
(0 45 t 0.041
167 t 39
(0.51 t 0.131
162 f 17
(0.36 f 0.05)
213 f 36
(0.39 f 0.091
168 2 13
(0.34 f 0.031
278 f 46
(0.36 t 0.04)
238 t 45
(0.37 t 0.03)
378 t 89
(0.33 f 0.041
407 2 80
(0.26 t 0.031
434 t 60
(0.25 t 0.041
542 t 45
(0.22 t 0.021
542 t 64
(0.19 t 0.02)
438 f 33
(0.17 t 0.011
545 c 80
(0.13 t 0.02)
347 t 191
(0.13 t 0.011
383 t 21
(0.78 t 0.05)
400 f 60
(0.91 ? 0.05)
342 t 60
(0.98 t 0.17)
738 t 164
(2.16 t 0.251
929 t 214
(1.98 t 0.41)
1001 t 159
(1.72 t 0.181
1202 t 198
(2 43 t 0.351
1161 t 229
(1.49 t 0.25)
1581 t 738
(2.15 t 0.77)
3095 t 333
(2.82 t 0.201
3840 t 432
(2.54 t 0.29)
3929 t 531
(2.20 t 0.131
4679 t 436
(1.87 t 0.15)
5247 t 559
(1.85 t 0.091
5487 t 725
(2.09 t 0.191
9320 f 1718
(2.21 t 0.401
5895 t 611
(2.42 t 0.4)
+
Rough
endoplasmic
reticulum
Outer
mitochondrial
membrane
Inner
mitochondrial
membrane
associated
vesicles
Lysosome
84 t 19
(0.17 t 0.041
73 t 17
10 17 f 0.031
109 f 22
(0.31 f 0.041
81 f 9
(0.26 t 0.051
139 t 44
(0.28 t 0.071
90 2 20
(0.15 t 0 031
84 t 18
(0.17 t 0.04)
240 t 21
(0.32 t 0.03)
161 t 52
(0.25 t 0.05)
423 t 33
(0.40 t 0.051
586 t 44
(0.39 t 0.06)
485 t 99
(0.28 t 0.061
650 t 102
(0.26 t 0.03)
591 t 124
(0.21 t 0.041
443 t 67
(0.17 t 0.031
913 t 158
(0.22 t 0.041
426 t 34
(0.17 t 0.021
117 t 30
(0.23 t 0.061
77 t 12
10.18 t 0.02)
113f 23
(0.31 t 0.04)
118 f 19
(0.35 f 0.04)
153 t 46
(0.31 t 0.071
185 2 33
(0.32 t 0.05)
131 t 17
(0.27 t 0.031
188 t 47
(0.24 t 0.061
243 t 78
(0.32 t 0.08)
633 t 174
(0.56 t 0.151
917 t 125
(0.60 t 0.061
998 2 109
(0.59 t 0.12)
1574 t 168
(0.63 t 0.071
1698 2 203
(0.60 t 0.061
1351 t 111
(0.53 t 0.08)
1542 t 197
(0.37 t 0.04)
970 t 71
(0.39 t 0.011
401 t 90
(0.80 t 0.151
211 t 15
(0.49 t 0.02)
278 f 45
(0.78 t 0.081
640 t 130
(1.89 f 0.221
753 f 221
(1.52 2 0.301
869 2 120
(1.52 t 0.181
611 f 51
(1.26 t 0.151
786 t 231
(1.00 t 0.291
894 t 338
(1.16 t 0.371
2446 t 578
(2.19 t 0.46)
3621 t 495
(2.37 t 0.301
4289 t 608
(2.51 2 0.531
3717 t 480
(1.49 t 0.20)
4232 t 555
(1.50 t 0.181
4547 t 404
(1.78 t 0.261
6476 t 969
(1.54 t 0.23)
1650 2 131
(0.67 t 0.071
122 t 56
(0.23 t 0.10)
38 t 12
(0.09 t 0.031
108 t 13
(0.30 f 0.031
103 f 71
(0.36 f 0.261
537 t 92
1.13 f 0.091
491 2 366
(0.88 t 0.631
408 t 173
(0.80 t 0.31)
602 t 223
(0.82 t 0.291
431 t 390
(0.54 t 0.491
430 t 430
(0.28 2 0.281
2045 t 994
11.30 t 0.58)
1652 t 371
(1.00 2 0.31)
2234 t 427
(0.92 t 0.20)
1938 t 620
(0.70 t 0.24)
1623 t 537
(0.65 t 0.26)
2723 t 1004
(0.63 2 0.231
377 f 125
(0.15 2 0.05)
9 t 3
(0.02 t 0.007)
523
(0.01 f 0.008)
7 t 1
(0.02 f 0.0031
18 f 4
(0.06 f 0.011
10 f 7
(0.02 f 0.011
34 t 11
(0.06 t 0.021
10 t 6
(0.02 t 0.011
322
(0.003 t 0.002)
11 t 9
(0.01 t 0.011
727
(0.008 t 0.008)
56 t 19
(0 04 -C 0 011
32 f 6
(0 02 f 0.0041
37 t 3
(0.02 t 0.0011
59 t 19
(0.02 t 0.006)
31t8
(0.01 t 0.0041
55 t 13
(0.01 t 0.003)
42 t 6
(0.02 f 0.0021
Discoid
Vesicles
associated
with
chromatoidlike body
I f 1
(0.004 t 0.0041
121
(0.002 2 0.0021
14 t 4
(0.03 t 0.0061
424
(0.004 t 0.0041
38 t 7
(0.06 t 0.01)
72 f 31
(0.07 2 0.03)
89 f 23
(0.06 t 0.01)
106 f 15
(0.06 f 0.011
34 f 16
(0.02 f 0.0071
18 t 8
(0.006 t 0.021
12 t 5
(0.005 t 0.003)
423
(0.001 f 0.0006)
115 t 47
(0.04 t 0.021
61 f 23
(0.03 t 0.01)
150 f 62
(0.04 t 0.01)
36 t 22
(0.01 t 0.007)
'Calculated for 2 animals since only 2 animals showed meiotic cells with nuclear membrane.
nide preparation. Occasional patches of ribosomes were
seen on endoplasmic reticulum that has the characteristics of the smooth variety (Fig. 15). For the preceding
reasons, smooth and rough components of the endoplasmic reticulum were considered as one during the spermiogenesis phase of spermatogenesis.
The surface area of the rough endoplasmic reticulum
(Fig. 4)remained constant during early spermatogenesis until zygotene (stages XII-XIII), where a n approximate 10-fold increase occurred until diplotene. The
surface area of rough endoplasmic reticulum was approximately halved during the first meiotic division.
The volume and surface area of the rough endoplasmic
reticulum showed similar trends in spermatogonia and
spermatocytes. Although rough endoplasmic reticulum
was not distinguished for quantitation in spermatids, i t
was our impression that fewer areas of rough reticulum
were seen a s spermiogenesis progressed.
Radial Body
The radial body (Fig. 16) was noted only in steps 8
through 18 spermatids. From step 8, both the volume
and surface area of this structure increased and then
decreased, providing a n overall pattern of a bellshaped curve with the peak at step 14 (Fig. 4).
Mitochondria
The pattern of the surface area of outer mitochondrial membrane generally paralleled that of the volume of mitochondria (Fig. 5). Outer mitochondrial
membrane surface area remained fairly constant in
cells up to preleptotene spermatocytes of stage VII.
From this cell type, the surface area increased such
that a 13-fold greater surface area was recorded up
through diplotene spermatocytes. In the subsequent
meiotic divisions, the surface area per cell decreased by
37% and 36% as compared with the predecessor celldiplotene spermatocyte and secondary spermatocyte,
respectively. The mean surface area of mitochondria as
expressed on a per cell basis remained relatively constant throughout spermiogenesis. The outer mitochondrial membrane surface area in the residual body was
about 16% of that in the step 19 spermatid (in stage
VIII).
The surface area of inner mitochondrial membrane
increased progressively from intermediate spermatogonia into diplotene spermatocytes, showing a n overall
approximate 31-fold increase during the time span that
these cells were present (Fig. 5). A dramatic decrease
(75%) in inner mitochondrial membrane occurred in
secondary spermatocytes. From secondary spermatocytes, there was a n approximate halving of this mitochondrial component in step 1 spermatids. No major
trends in surface area of inner mitochondrial membrane were noted throughout spermiogenesis.
Golgi Apparatus
Considerable variability in the data from the surface
area determinations of Golgi apparatus were apparent,
given sampling problems posed by a n organelle concentrated a t one site within the cell and thus only occasionally encountered in sectioned material. Trends in
u
In
..a,.
””...
In
2550
PlDl
B
s
-
187
I i...
19m
..... ......
.*....,...,....
[ m ) ll-lV[m)
1
GERMCELLMORPHOMETRY
2
4
V
5
e
7
v
n
VII
8
ell
12-13 14
15
1617
17
18
19
19
II-IV
v
vl
vll
vlll
3ooo
2.m
2253
=
- ztm
m
ieso
-5
1-
2
1=
l?m
2 1Qo
E
15m
g 1-
* 9 m
7Y)
Qo
450
300
150
0
Stage
I
CIV
VUI IX-XI XCXUl
XIV
I
Figs. 1-10, Line graphs showing volume and surface area parameters for various germ cell types. In most instances, the top graph
shows spermatogonia and spermatocytes and the bottom graph only
spermatids. Abbreviation used: Roman numerals I-XIV are the
stages of the cycle of the seminiferous epithelium; V, volume; SA,
surface area; A, type A spermatogonia; In, intermediate spermatogonia; B, type B spermatogonia; P1, preleptotene spermatocytes; L,
leptotene spermatocytes; Z, zygotene spermatocytes; P, pachytene
spermatocytes; Di, diplotene spermatocytes; S, secondary spermatocytes; Arabic numerals 1-19, steps of spermiogenesis. The numerical
scale for figures for a particular parameter may not be similar for on
the top and bottom graphs. Line graphs were made using a “best fit”
curve employing the fourth polynomial in regression analysis
(Statview I1 for Macintosh). Dotted lines representing volumes or surface areas indicate that cell divisions (m) take place in the periods
examined whereas solid lines indicate continuous cell development
without cell division. Separations between stages are roughly proportional to the length of those stages.
volume and surface area of the Golgi were similar
throughout spermatogenesis (Fig. 6). The Golgi in spermatogonia through stage VII preleptotene spermatocytes showed no surface area or volume trends. However, in preleptotene cells a t stage VIII Golgi surface
area appeared to increase progressively until the
diplotene phase of meiosis.
There was a marked (86%) reduction in the surface
area of Golgi in secondary spermatocytes (Fig. 6). Although cell division occurred to produce step l spermatids, the surface area of the Golgi of newly formed spermatids was similar to that of secondary spermatocytes.
A slight trend for increase in the surface area of the
Golgi was noted in spermiogenesis until step 8, where
the trend reversed. The surface area of the Golgi declined until step 19 (in stage VII), where no Golgi apparatus, or elements thereof, were identified. Golgi, a s
defined as the membrane sacs inside the presumptive
cytoplasmic droplets of spermatids/sperm (Oko et al.,
19931, was seen in step 19 spermatids (in stage VIII)
and in the residual body where their surface area was
recorded.
It was suggested by the quantitative data and verified by visual inspection of the Golgi that the volume of
saccular elements of the Golgi apparatus decreased
during spermiogenesis. Some micrographs show apparent dissociation of the Golgi and walling off of some
portions of the Golgi (Fig. 18).
Fig. 1. Cell and plasma membrane.
Lysosomes and Multivesicular Bodies
Lysosomes were identified morphologically and not
by demonstration of acid hydrolases. Using this criteria, there was little evidence of a trend throughout the
spermatogenic cycle (Fig. 6). Pachytene cells in stage V
through secondary spermatocytes appeared to show increased lysosomal volume as expressed on a per cell
basis a s compared with less mature germ cell types.
Spermatid lysosomes were less numerous on a per cell
basis than in pachytene spermatocytes. A decline in
lysosomes occurred in step 18 and 19 spermatids.
188
L.R.
DE
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TABLE 5. Spermatids surface parameters (mean
2
SE);
Golgi
Step
Or
stage
1
I
2-4
11-IV
5
V
6
VI
7
VII
8
VIII
9-11
IX-XI
12-13
xII-xIII
14
XIV
15
I
16-17
11-IV
17
V
18
VI
19
VII
19'
VIII
Residual'
body VIII
'N
'N
Plasma
membrane
Nuclear
membrane
926 f 107
(0.72 f 0.041
959 f 50
(0.67 f 0 05)
1256 f 156
(0.74 f 0.061
1372 f 62
(0 73 f 0.051
1138 f 47
(0.60 f 0.021
1328 f 198
(0 87 f 0.071
1695 f 218
(1.33 t 0.171
1755 f 261
(1.50 f 0.081
1733 f 122
(1.47 2 0.091
1876 f 93
(1.59 f 0 121
1847 f 170
11.49 f 0.201
1780 f 304
(1.61 f 0.141
1832 f 306
(2.06 f 0.311
1339 f 280
(2 33 f 0.14)
1436 f 460
(3 06 f 0.251
256 f 27
(0 87 f 0 131
308 f 46
(0.24 2 0 021
279 t 24
1019f0011
370 2 50
(0.22 f 0.011
370 f 80
(0.19 f 0.031
392 f 16
(0.21 f 0.011
305 f 55
10.20 f 0.03)
209 f 56
(0.16 f 0.041
211 f 47
(0.19 f 0.05)
94 f 24
(0.08 f 0 03)
105 f 21
(0 09 t 0.011
125 2 44
(0.11 f 0.041
76 f 22
(0.07 f 0.021
147 f 62
(0.17 f 0.071
92 f 33
(0.15 f 0.041
49 f 7
(0.11 f 0.011
Acrosomic
membrane
Endoplasmic
reticulum
24 t 19
(0.02 t 0.01)
64 t 16
(0.04 f 0.011
144 f 45
10.07 f 0.02)
286 f 26
10.15 f 0.01)
161 f 21
(0.11 f 0.011
277 f 72
(0.22 f 0.061
277 f 87
(0 26 f 0.09)
214 f 52
(0.19 f 0.05)
246 f 33
(0 20 t 0.021
272 2 90
(0.22 2 0.081
145 f 57
(0.13 f 0.061
276 f 106
10.32 f 0.12)
245 f 127
(0.39 f 0.151
191 f 57
(0.41 ? 0.051
2992 f 640
(2 33 f 0 451
3382 f 281
(2 34 f 0 141
4278 f 806
(2.51 f 0.301
4520 2 528
(2.36 2 0.161
3454 2 205
(1.81 2 0.101
3667 2 482
(2.40 2 0.19)
3736 2 355
(2.92 2 0.27)
3552 f 580
13.00 f 0.171
3189 f 373
(2 69 f 0.27)
3311 2 636
(2.59 f 0.51)
2861 t 289
(2.26 f 0 131
2286 f 447
(2.04 2 0.091
1263 f 189
(1.40 2 0 131
200 2 52
(0.34 2 0 041
180 2 57
(0.39 f 0.081
26 f 5
10.09 f 0 02)
Radial
body
42 f 25
(0.03 f 0.021
140 f 99
(0.11 2 0.071
189 f 92
(0.14 f 0.071
223 f 78
(0 19 f 0.061
205 f 99
(0.16 f 0.071
93 f 48
(0.07 f 0.041
144 f 21
(0.15 f 0.03)
56 f 19
(0.09 f 0.031
Outer
mitochondrial
membrane
Inner
mitochondrial
membrane
618 f 78
(0.48 f 0 041
776 f 80
(0 54 f 0 041
916 f 70
10.55 f 0.05)
1040 f 73
10.55 f 0.031
728 2 20
(0.38 f 0.01)
886 f 139
(0.58 t 0.061
726 t 76
(0.57 f 0.07)
745 f 105
(0.63 f 0.031
695 f 77
(0.59 f 0.05)
801 f 68
(0 68 f 0.061
627 f 65
(0.51 f 0.07)
737 t 168
(0.66 f 0.071
611 f 148
(0.67 f 0.131
627 f 205
(1.08 f 0.231
684 f 240
(1.45 f 0.131
111 f 2
(0.37 f 0.02)
855 f 169
(0.66 f 0 091
999 ? 85
(0.69 f 0 051
1118 f 105
(0 67 f 0.071
1281 t 59
(0 68 f 0.041
984 2 29
(0.52 f 0.02)
1275 f 200
(0.83 f 0.091
1279 f 143
(1.10 f 0.131
1310 f 191
11.11 f 0.05)
1202 f 130
(1.01 f 0.051
1351 f 131
(1 14 t 0.121
1089 t 96
(0.88 f 0.111
1213 f 325
11.06 f 0.141
891 f 226
(0 97 f 0.20)
901 f 289
(1.55 f 0 321
757 f 267
(1.60 f 0.151
113 f 2
(0 38 f 0 02)
+
associated
vesicles
387 f 102
(0.29 t 0.061
349 t 107
(0.24 t 0.07)
539 t 160
(0 34 f 0.121
405 f 91
10.21 f 0.041
523 f 130
(0.27 f 0.061
747 f 157
(0.49 f 0.081
627 f 83
(0.49 f 0.061
503 f 154
(0.42 f 0.101
532 f 71
(0.46 f 0.081
466 f 87
(0 38 f 0.051
265 f 138
(0.20 f 0.101
168 f 42
(0.17 f 0.05)
24 f 14
(0.03 f 0.021
Lysosome
14 f 3
(0.01 f 0.002)
22 f 6
10.02 f 0 005)
28 f 11
(0.02 f 0 005)
18 2 4
10 009 2 0.001)
8 2 1.3
(0.004 f 0.0011
12 f 2.3
(0.008 2 0.0011
6.8 2 3
(0.006 2 0.0021
14 2 8
(0.02 f 0.0081
32 2 8
(0.03 f 0.0071
555
10 005 f 0 0051
48 2 12
(0 04 2 0.011
14 2 6
10.01 2 0 0041
929
(0.01 f 0.01)
2.4 2 1.4
(0 004 2 0.0031
112 t 67
(0 21 f 0.081
20 2 10
(0 07 f 0 04)
= 3.
= 2.
Lysosomes with classical morphological features
were not seen in residual bodies although reports of
acid phosphatase activity in vesicular elements have
been published (Bozzola and Russell, 1992; Chemes,
1986).
Multivesicular bodies were only observed in spermatids (Tables 3 and 5). Their numbers were infrequent
and thus the stage-related variability in volume and
surface area was great. However, the volume and surface area parameters appeared to selectively increase
in step 16, 17 and 19 spermatids. The residual body
exclusively contained the multivesicular bodies since
none were seen in sperm (Fig. 11).
Acrosome and Subacrosomal Space
The acrosome increased in volume until step 7 of
spermiogenesis and thereafter slowly decreased its volume until the completion of spermiogenesis such that
the step 19 spermatid acrosome was about 29% of its
maximal size (Fig. 7).
The surface area of the acrosome (Fig. 7) generally
paralleled the increase in volume until step 7, where i t
continued to increase a s the acrosome spread over the
nucleus during steps 8 through 12. Thereafter during
spermiogenesis the acrosomal surface area remained
relatively stable.
The subacrosomal space (Fig. 7) gradually increased
in volume during spermiogenesis until steps 12-13,
where a slight pattern of decline was noted that lasted
until step 19. At step 19, the subacrosomal space was
only 32% of its peak size.
Lipid
Lipid was not seen until mid-pachytene, although it
was sparse in this cell type. In spermatids (Fig. 8), lipid
increased progressively during spermiogenesis until
step 18 where its volume stabilized. Lipid was recorded
to be present only in the residual body and not in the
step 19 sperm that were separated from their residual
cytoplasm (Fig. 11).
Outer Dense Fibers and Fibrous Ring
Outer dense fibers and fibrous ring material were
first noted at the magnifications used in steps 9
through 11 spermatids. Their combined volume increased dramatically (16-fold) throughout spermiogenesis (Fig. 8).
Vesicles
Vesicular elements were a major component of spermatids, especially elongating and elongate spermatids.
Vesicles have not previously been classified by morphological or functional (if any) criteria. They were herein
defined by morphological criteria or by associated
structures. The various types of vesicles are illustrated
(Figs. 17-23).
Endoplasmic reticulum-associated vesicles (Figs. 9
and 17) were first seen in step 9-11 spermatids and
were characterized by the presence of endoplasmic reticulum aligned in single file (as seen in sections) outside the bounding membrane of the vesicle. Endoplasmic reticulum-associated vesicles increased to reach
their peak volume in step 12-13 spermatids. From
steps 12 to 13, their volume decreased such that they
could no longer be identified by the aforementioned
criteria in step 19 spermatids a t stage VIII.
Small vesicular elements associated with the chromatoid body (Fig. 19) were first seen in stage VIII
189
G E R M CELL MORPHOMETRY
surface area (pm2)and surface density in parentheses
Multivesicular
body
19213
(0 002 2 0.001)
09 f09
(0.001 2 0.001l
2022
(0.002 f 0.002)
7 8 f 4.7
(0.004 f 0.002)
9253
(0.005 2 0.003)
3.3 f 2
(0.002 f 0.001)
2.9 f 1.7
(0.003 f 0.001)
1.9 f 1.9
(0.002 f 0.0021
2.2 f 1.4
10.002 f 0.0011
10 f 3.4
(0.008 f 0.0031
12.5 t 1 8
(0.028 f 0.011
14.3 f 8.7
(0.04 f 0.021
9?9
(0.03 t 0 031
Vesicles
associated
with chromatoid body
95 2 22
(0.07 2 0.02)
50 2 14
(0.03 2 0.01)
82 2 28
(0.05 2 0.011
83 2 17
(0.05 2 0.01)
35 2 10
(0.02 2 0.005)
52 2 16
(0.04 2 0.011
59 f 18
(0.05 2 0.011
60 2 15
(0.05 2 0.011
23 2 10
(0.02 r 0.011
18 f 3
(0.02 f 0.004)
Myelin
figure
Clear vesicles
associated with
endoplasmic
reticulum
Clear
associated
vesicles
V e s ic1e s
associated
with dense
material
Lightly
granular
vesicles
Vesicles
with
medium
density
Endoplasmic
reticulum
close to
manchette
Tubulobulbar
complex
44 f 9
(0 03 f 0.0011
25 f 16
10.01 f 0.007l
21 t 7
(0.01f 0.004)
6.8 f 2.5
(0.003 f 0.001)
121
(0.001 ? 0.001l
*
9.9 5 7
(0.008 ? 0.005)
16 ? 9
(0.01 t 0 006)
0.8 t 0.8
(0.001 2 0.001l
3522
(0.003 ? 0.0021
1.9 2 1.9
(0.002 ? 0 0021
3.2 2
(0.002 2
50 2
(0.04 2
136 2
(0.12 2
185 2
(0.16 2
167 2
(0.14 2
23 2
10.02 2
11 2
(0.01 2
22 2
(0.02 2
51 2
(0 08 2
2.1
0.001l
23
0.021
49
0.04)
21
0.011
59
0.04)
5
0.004)
7
0.0081
10
0.0091
22
0.02)
35 2 12
(0 03 f 0 005)
84 f 16
10.01 f 0.021
34 2 14
10.03 ? 0.001)
58 f 23
(0.04 f 0.02)
57 f 24
(0 05 f 0 021
25 f 9
10.03 f 0.0081
1.8 t 1.8
10.001 2 0.001)
19 t 15
(0.01 t 0.01)
20 2 9
10.02 t 0.006)
26 2 3
10.02 2 0.002)
90 2 36
(0.07 2 0.03)
49 2 16
(0.04 2 0.01)
281 2 93
(0.28 t 0.121
45 t 45
(0.03 t 0 031
161 t 62
(0.29 2 0.09)
207 2 45
(0 48 2 0.06)
176 2 2
(0 59 2 0.011
Fig. 2. Nuclear membrane, ground substance and nucleus.
2 4 2 11
(0.05 2 0.021
21 t 7
(0.05 f 0 02)
14 f 6
(0.05 f 0.021
28 f 11
(0.05 f 0.01)
190
L.R.
DE
FRANCA ET AL.
mn.
750
-5m
200
Stage
I
w
v
VI
VI
VII
Ix-XI XIMM
m
I
M
v
VI
VI
-
150
L
O
vni
Fig 3 Smooth endoplasmic reticulum (top graph) and total endoplasmic reticulum (bottom graph)
pachytene spermatocytes. In step 1-14 spermatids, the above. Vesicles with a granular interior and small vesvolume of these vesicles remained relatively constant icles of moderate density showed occasional intermedi(Fig. 10).At step 15 there was a sharp decrease in these ate forms.
parameters. Vesicles of this type were not noted thereTubulobulbar Complexes
after.
Tubulobulbar complexes were recorded only in step
Clear vesicles (not associated with endoplasmic reticulum; Fig. 9, Fig. 20) were first seen in stages 12-13. 19 spermatids of stage VII (Table 3).
Their volume was relatively constant until step 18,
Discoids
where i t declined, not to be detected in subsequent
Structures that were membrane bound and which
stages. Clear vesicles were normally situated in the
caudal cytoplasm (Fig. 17). In step 14, there were nu- contained dense material with translucent areas for a
merous clear vesicles containing myelin figures. Occa- better lack of a name were called “discoids” due to their
sional images were obtained showing the bounding apparent discoidal shape (Fig. 24). Discoids were first
membrane of clear vesicles in continuity with endo- noted in leptotene cells of stages IX-XI and increased
to reach peak volume in stage VI pachytene cells. Their
plasmic reticulum (Fig. 22).
Vesicles associated with dense material (Figs. 9, volume decreased thereafter in primary spermatocytes,
16C, 18, 21) were first identified in step 9-11 sperma- not to be seen in secondary spermatocytes and more
tids. They were detected until step 18 of spermiogene- mature cells (Fig. 10).
sis. They attained a peak volume in steps 16-17 (in
Nuage and Associated Vesicles
stages 11-IV). Aggregations of these vesicles were norThe term nuage is used to describe a variety of dense
mally situated near the spermatid plasma membane.
In the later steps of spermiogenesis these vesicular el- materials within germ cells (Russell and Frank, 1978).
It is a general term used herein to describe and to
ements appeared to flatten (Figs. 18, 22).
Vesicles with a granular interior (Fig. 22) and small quantify a t least five forms of dense material in spervesicles of moderate density (Fig. 23) were only noted matocytes. However, in spermatids dense material was
in step 19 spermatids. Not all of these vesicles had categorized into three types for quantitation: the chrodistinct morphology in step 19 spermatids but most matoid body, the reticulated body, and granulated body
could be characterized as one of the two types described (Clermont et al., 1990; Fawcett et al., 1970).
191
GERM CELL MORPHOMETRY
B
B
P
I
P I L
lA2
,,..
.....
V
ym)
..........
- .... .....
VII
vln IX-
Fig. 4. Rough endoplasmic reticulum (top) and radial body (bottom).
Nuage (Fig. 10) was first recorded in the present
study in leptotene spermatocytes where it appeared to
increase progressively in amount (volume), attaining a
peak volume in diplotene phase of meiosis.
In spermatids, the chromatoid body (Fig. 10)progressively decreased in volume until step 14. It was not
seen thereafter.
The granulated body (Figs. 10, 17) was first seen in
steps 12-13 and increased in volume until step 17. Step
19 spermatids contained little material distinguished
as the granulated body.
The reticulated body (Figs. 10, 17) was first seen in
step 14 and was most abundant in step 15 spermatids.
Steps 16-18 showed few reticulated bodies.
Myelin Figures
Myelin figures were noted within the cytoplasm of
the spermatid population of cells beginning a t steps
2-4 of spermiogenesis. They were not seen past step 17
of spermiogenesis (Fig. 25).
Composition of Spermatids Separated From the Residual
Body and the Residual Body
Data for the volumetric composition of sperm and the
residual bodies are shown (Fig. 11).The head of the
sperm comprised about 10.4% of its volume which rep-
resented a slightly greater volume than the cytoplasmic droplet.
Percentage of Flagelia in Lumen
In the course of obtaining this data we calculated the
percentage of the spermatid that extended into the lumen. The lumen was defined as that portion of the
tubule that was more apically situated than the apical
tips of the Sertoli cell processes. In each instance, the
percentages given represent the flagellar protrusions
of spermatids. For each spermatid cell type the percentages are as follows: step 1,0.0%; step 2-4,0.3%; step 5,
0.5%;step 6,0.6%; step 7,0.7%; step 8, 1.3%; step 9-11,
1.8%; step 12-13, 2.2%; step 14, 1.4%; step 15, 2.3%;
step 16-17 in stages I1 to IV, 1.8%; step 17 in stage V,
1.4%; step 18,2.6%;step 19 in stage VII, 17.8%;step 19
in stage VIII, 46.5%.
DISCUSSION
A comprehensive measurement of volumes and surface areas necessitates that all cell components be
quantified using either the line intersection or point
counting methods. Thus, we encountered previously
unnamedlundescribed structures which dictated that
names be given to them for segregation of quantitative
data. New observations on previously described structures are also provided.
VlI
VHI IX-XI
XII-XU1 XIV
I
Fig. 5. Mitochondria and outer and inner mitochondria1 membranes.
Stage
I
nw
v
n
vn
nn
IX-XI XWXII
XN
I
IHV
v
Fig 6 Golgi and lysosomes
n
w
nn
193
GERMCELLMORPHOMETRY
5
300
8
8
7
9-11 12-13 14
15
1617 17
285
3 120
105
SO
45
30
15
IUV
I
Stage
Fig. 7. Acrosome and subacrosomal space.
8
v
vl
VII
3-11
12-13 14
vlll I X - n X M l l l
15
I
XIV
1617
17
18
IUV
v
VI
19
VII
19
Vlll
Stage
Fig. 8. Lipid and outer dense fibers plus fibrous ring.
-11
12-13 14
15
1617
17
18
19
v
VI
vll
19
I I l l /
I
IUV
v
vl
vlI
ill I X - i l XlCXlll
av
I
WV
Ylll
Stage
Fig. 9. Endoplasmic reticulum (ER)-associatedvesicles and clear vesicles and vesicles associated with
dense material.
194
L.R.
B
B
DE
A
FRANCA ET AL.
Nj_
V
I
IHV
14
15
1617
P
P-DI
s
I t
/
.XI XlCXlll
1
24
5
6
7
8
C11
12-13
17
18
19
10
Fig. 10. Nuage, discoids (top),chromatoid body, chromatoid associated vesicles, granulated body and
reticulated body (below).
The present study shows temporal relationships in
the presence and absence of particular structures that,
in some instances, correlates with the presence and
absence of other structural entities. There are many
possible inferences that can be made about the temporal relationships of the appearance and disappearance
of structural entities. Generally, we avoided doing so
because this study did not target all time periods during spermatogenesis since i t was constrained by the
process of random sampling. Admittedly, the present
study has raised numerous questions about structural
relationships of various cell constituents and about
these constituents and functional changes that occur
during the spermatogenic cycle. For the most part we
desired to avoid speculation and to leave unresolved
questions to be answered in future research. There is,
however, information that strongly argues for linking
structural relationships or for correlating structure
with function in a temporal sense. It is the more wellfounded observations that will constitute the bulk of
the discussion that follows.
Cell Parameters
Examination of Figure 1 shows nicely how cell volume and cell surface area data parallel each other for
most of early spermatogenesis. However, the chart also
shows a change in relationship of surface area and volume beginning a t the elongation phase of spermiogenesis (step 8). This is caused by a n increase in cell surface area and a decrease in cell volume. Cell volume
decreases in spermatids have been described, especially late in spermiogenesis (step 19) and the cause for
this specific volume decrease discussed (Roosen-Runge,
1955; Russell, 1979,1980). However, the present study
is the first to pinpoint the volume decreases to steps
8-11 of spermiogenesis.
There are dramatic fluctuations in cell volume during spermatogenesis. One cell that began as Type A
spermatogonia with a mean volhme of about 492 pm3
yielded 4,096 spermatids attached to a cytoplasmic lobe
(theoretical yield without considering cell degeneration; Ren and Russell, 1991), each with a mean volume
of 452 pm3. During the developmental process germ
cells reaches a maximum size of 4,202 pm3 (diplotene).
Each spermatid released as a sperm was about 166 pm3
in volume, and left behind 298 pm3 of cytoplasm that
the Sertoli cell phagocytosed. Considering the sperm
production in the r a t as being 36 million per day per
paired testis (Russell and Peterson, 1984), the Sertoli
cell engulfed and dimosed of 10.1mm3 of cvtodasm
" .
-
195
GERMCELLMORPHOMETRY
A. Spermafid w/o Residual Body
766.2pW
8. Residual Body
298 p H
'Golgi"
2.7prn3
0 9%
Lipid
19.0prn3
6.4% \
7 Mitochondria
}
10.7pm3
GKound5ilhhm3
3.6%
186.3urn3
62.6Y0
Mutltivesicular Body
4.0prn3
1.3%
Subacrosornal Space
Sperm Head
17.1prn3
10.4%
Sperm Flagellum
(including cytoplasmic droplet)
149.1 prn3
89.6%
Cytoplasmic droplet only
12.3vrn3
7.4%
Fig. 11. Diagram showing the components of a rat spermatid a t late step 19 of spermiogenesis that has
not been released from the Sertoli cell but has been disengaged from the residual body. The absolute
volumes and the percentage volume occupancy of the spermatid and the residual body are provided.
from released sperm as the residual body! This daily final position segregating the midpiece from the reelimination represented about 0.3% of the mass of each mainder of the flagellum in late step 15. This movetestis. In approximately one year the Sertoli cells ment eliminated what may be called the flagellar cawould phagocytose residual bodies to the equivalence nal. Since there is not apparent decrease in cell surface
area at this time, i t is assumed that the plasma memof the weight of both testes.
Data from the present study was in general agree- brane surface area is displaced from around the flagelment with a previous study on the surface relation- lum rather than resorbed by this process.
Cell growth, a s reflected by cell volume, increased
ships of Sertoli cells (FranCa et al., 1993). The curves
for Sertoli surface area and germ cell surface area dramatically in pachytene spermatocytes, especially a t
match closely throughout spermatogenesis suggesting mid-pachytene in line with previous observations (Rusthat the Sertoli and germ cell established relationships sell and Frank, 1978a). The nuclear volume increased
with one another based on the surface areas of one or in this period but there was a much greater increase in
both cells. Data from developing animals and from Ser- the ground substance. The great increase in ground
toli cells in culture (Kelly et al., 1991) and models substance was assumed to be necessary in anticipation
where germ cells are depleted (Ghosh et al., 1992a,b; of the two rapid cytokineses occurring in stage XIV,
Sinha Hikim et al., 1989) indicate that the Sertoli cell which would essentially divide the cell into four cells
responds by increasing its surface area secondary to given little time for synthetic processes to occur.
During spermiogenesis, the nuclear volume declined
the presence of germ cells.
The annulus moves from the nucleus toward to its sharply from step 8 through step 14, most likely due to
Figs. 12-15
GERMCELLMORPHOMETRY
197
Fig. 16. The radial body a t three phases of development. In A (step 14), the radial body contains
saccular elements that show a clear lumen. In B (step 151, elements of the radial body are short and
display a less apparent lumen than that shown in A. In C (step 18), the radial body appears to be
disintegrating. All x 23,400.
Fig. 12. Nuclear invaginations in late step 7 spermatids (A, B). The
nuclear envelope (arrow)takes an irregular course after invaginating
the nucleoplasm. Cytoplasmic constituents do not enter the invaginated region as evidenced by the clear (c) region. Both x 36,000.
Fig. 13. Large saccules of endoplasmic reticulum. In A, the endoplasmic reticulum (arrowhead) of a step 12 spermatids is in saccular
form and largely free of ribosomes. It is depicted in a position typically
flanking the manchette microtubules. In B (late step 12), a t a time
when saccules are lacking in a position flanking the manchette, saccules of endoplasmic reticulum such as that depicted (arrowhead) can
be found isolated within the cytoplasm. Both x 36,000.
Fig. 14. Endoplasmic reticulum. Two forms of endoplasmic reticulum can be seen in this micrograph of a step 16 spermatid. One (arrowheads) is more typical of that seen at most steps of spermiogenesis.
The other (arrows) is composed of small saccules of endoplasmic reticulum whose walls are relatively collapsed and parallel. x 18,000.
Fig. 15. Patches of rough endoplasmic reticulum (arrowheads) are
noted among typical appearing smooth endoplasmic reticulum in this
step 2 spermatid. x 27,000.
Fig. 17. Survey micrograph showing numerous cell constituents in a step 15 spermatid. Indicated are
the following: flagellar canal lined by endoplasmic reticulum (open arrows) on the cytoplasmic side;
endoplasmic reticulum-associated vesicles (dark arrows); aggregations of clear vesicles (v); granulated
body (gb); reticulated body (rb); radial body (r);Golgi apparatus (G); lipid (1) and penetrating processes
(pp) of Sertoli cell. x 7,500.
199
GERMCELLMORPHOMETRY
Fig. 18. Golgi in step 17 of spermiogenesis. The Golgi (G) shown is
small. A portion of the Golgi is walled off within a membrane sac
(arrowheads). Also shown are vesicles with associated dense material
(arrows). x 36,000.
Fig. 19. A Small vesicles associated with chromatoid body in a step
2 spermatid. B The small vesicular element associated with the chromatoid body are fewer, but larger, in this step 9 spermatid. Indicated
are lysosomes (l), a myeling figure (mf) and multivesicular bodies
(mv). x 27,000; x 23,400, respectively.
nuclear condensation. At the same time, the cell volume decreased without any detectable change in the
volume of the ground substance, suggesting that the
contents of the nucleus were not just simply transferred to the cytoplasm but were actually eliminated
from the cell. We suggest that the constituent eliminated was mainly water since the genomic properties of
the nucleus and associated nuclear binding proteins
are known to remain.
The marked decrease in cytoplasm that occurred late
in spermiogenesis a t step 19 has been related to the
process of phagocytosis of tubulobulbar complexes and
possibly continued elimination of water from the cell
and has been amply discussed (Russell, 1980, 1984,
1993).
Nuclear Parameters
Nuclear volume remained reasonably constant until
the preleptotene phase of spermatogenesis, where a
steady increase in volume occurred most likely to accommodate the spatial requirements of chromosome
pairing and crossing over. The nuclear volume was approximately halved in meiosis I, but the nucleus only
lost about a quarter of its volume subsequent to meiosis
11. Thus, the haploid nucleus is actually larger than
that of diploid spermatogonia. The nucleus appeared to
200
L.R.
DE
FRANCA ET AL.
Figs. 20-23.
201
GERM CELL MORPHOMETRY
Fig. 24. Discoids (arrowheads). Discoids may be sectioned across their flat surface (A and B) or in
parallel with their flat surface (C).Most discoids show both a dense and translucent core (B and C).Some
discoids show little evidence of the electron translucent interior component. Some (A), may contain
membranous elements. All micrographs are from pachytene cells. Stages XIV, V, and XIV, respectively;
all ~27,000.
Fig. 25. Myelin figures. Two myelin figures (arrows) are shown within the cytoplasm of a step 4
spermatid. x 23,400.
grow in parallel with the cell in early spermiogenesis.
However, at step 8 its volume declined sharply until
step 14, a t which time its volume stabilized to about 4%
of its former volume, reflecting greater nuclear packaging that accompanied replacement of histones by
protamines.
One particular feature noted at late step 7 and a t
step 8 of spermiogenesis was the appearance of nuclear
invaginations that occupied 1.0-0.5%, respectively, of
the nuclear volume. In the past we and others have
attributed the presence of these unusual nuclear membranes to problems of fixation at these particular
stages. However, in light of the present data showing a
sharp drop in nuclear volume at these times, it is suggested that the nuclear surface area must rapidly accommodate a decreasing nuclear volume. Apparently
the nuclear envelope involutes into the nucleus and by
step 9 the involution has disappeared or has been reabsorbed.
Nuclear involution is not simply caused by a flow of
cytoplasm into the nuclear interior because no organelles or cytoplasmic constituents are seen in the
area of the invagination. One possible explanation for
the clear area observed in the invaginated pocket is
that water is extruded from the nucleus into this particular region and is in the process of transfer from the
nucleus to the cytoplasm.
Fig. 20.Clear vesicles. A typical aggregation of clear vesicles (arrows) is shown in a step 16 spermatid. The interior of such vesicles
sometimes appears dense but this dense appearance is the vesicular
wall sectioned en face. x 36,000.
Endoplasmic Reticulum
Fig.21.Vesicles associated with dense material. Both A and B (step
12) show small vesicular elements in association with dense material.
In both instances these vesicles adjoin the spermatid plasma membrane. x 23,400; x 27,000, respectively.
Fig. 22. Vesicles with granular interior. Large rounded vesicular
elements with a finely granular texture are shown in this step 19
(stage VII) spermatid (gv). In addition, a clear vesicle (v) with its
bounding membrane continuous with the endoplasmic reticulum (arrowheads) and vesicles associated with dense material (arrow) are
also shown. x 36,000.
Fig. 23. Small vesicles of moderate density. Small vesicles with a
content of moderate density are aggregated in the cytoplasm of this
step 19 spermatid (of stage VIII; arrows). Also shown are lipid (11,
mitochondria (m), multivesicular bodies (vb), endoplasmic reticulum
(er) and vesicles with a granular interior (gv). x 22,500.
In spermatogonia and spermatocytes, the volume
and surface area of the smooth endoplasmic reticulum
and rough endoplasmic reticulum increased in parallel.
The smooth endoplasmic reticulum was maintained a t
about 5-10-fold greater in amount and in surface area
than the rough endoplasmic reticulum. In spermatids,
occasional ribosomes were seen on endoplasmic reticulum that was non-saccular and had a n appearance
more like the smooth variety. Since these two forms of
endoplasmic reticulum could not be differentiated
readily they were considered together.
The data show that in spermatids from step 14
through 19 of spermiogenesis, the volume of the endoplasmic reticulum decreased less than its surface area,
leaving only the possibility that the endoplasmic reticulum assumed more of a rounded form in these sper-
202
L.R.
DE
FRANCA ET AL.
matids. We do not know if this represents a differential
response of endoplasmic reticulum to fixation conditions or represents a functional property of the smooth
endoplasmic reticulum.
In steps 12 to 13, the endoplasmic reticulum formed
large saccules flanking the developing manchette.
These were apparently lost from the region of the
manchette since in advanced steps 12-13, large saccules were observed scattered throughout the cytoplasm. In step 14, the endoplasmic reticulum flanking
the manchette is highly fenestrated (Clermont and
Rambourg, 1978), leading the investigator not to know
if the saccular form was replaced with the fenestrated
variety or modified from saccular to fenestrated endoplasmic reticulum.
The volume and surface area graphs indicate that
volume of the Golgi is decreasing more rapidly than its
surface area. This data can be reconciled by examination of electron micrographs showing a volume decrease in individual saccular elements later in spermiogenesis.
Mitochondria
Lipid
The volume density data for mitochondria indicated
that the occupancy of mitochondria changed little
throughout spermatogenesis. This organelle was the
most constant feature, in terms of its percentage occupancy of cells, as they divided and increased and decreased in size during spermatogenesis. Thus, in future
studies it may be used a s a reference to base the relative frequency of other structures in the cell.
The data pertinent to mitochondrial structural parameters in primary spermatocytes shows that the inner mitochondrial membrane surface area did not parallel that of the outer mitochondrial membrane. Since
the surface area of the inner membrane increased dramatically while the outer membrane shows only a
slight increase it indicates that the specific enzymecontaining components of the mitochondria were differentially increasing in surface area at mid and late
pachytene suggesting a n increased need for mitochondrial function at this time. No clear trend for mitochondria in this respect is noted in spermatids.
Lipid was not seen in spermatogonia and only rarely
noted in spermatocytes. From step 1of spermiogenesis
it increased in volume at a slow rate through step 14 of
spermiogenesis, but increased dramatically thereafter
until step 18 (in stage VI) when its volume subsequently leveled off. All lipid in the form of droplets is
eliminated in the residual body.
No function for lipid in spermatids has been proposed
in the literature, but the lipid build-up that does occur
likely represents degradation of germ cell products
rather than representing a storage precursor pool. That
tremendous amounts of membrane are being lost in
late spermiogenesis can be verified by examination of
graphs for cell surface (Fig. 11, endoplasmic reticulum
(Fig. 3), Golgi (Fig. 6), etc. Since unit membranes are
mostly lipid and lipid occupies a large part of the
phagocytosed residual body, i t may be that lipid represents the accumulated products of lipid metabolism by
the spermatids that is necessary prior to phagocytosis
of the residual body a s to not overwhelm the Sertoli cell
(metabolically) with unprocessed lipid precursors.
Golgi and Acrosome
Golgi volume and Golgi surface area increased linearly in spermatocytes to reflect the increased synthetic activity of these cells (Monesi, 1970). Growth of
the Golgi in early spermiogenesis as the acrosome is
formed provides a second indication of Golgi function.
Expectedly, the acrosome rapidly increases in volume
and surface area in early spermiogenesis to reach its
peak in both parameters in step 7 spermatids. Unexpectedly, while the surface area of the acrosome was
maintained thereafter (situated on a much smaller nucleus), the volume of this component steadily decreased
to approximately 1/3 of its peak value by step 19. The
significance of the volume decrease is not known.
The Golgi remains structurally elaborate a s exemplified by volume and surface area parameters until
steps 9-11, where both parameters declined, thereafter
not to be observed in step 19 spermatids at stage VII.
Since a marker Golgi enzyme is found within saccules
of the cytoplasmic droplet (Chan et al., 19901, we
herein consider the Golgi apparatus to reappear or reform from scattered cisternae in step 19 spermatids in
stage VIII and to reform in the residual body although
the structural components seen may not, in the future,
be traced as Golgi apparatus. Some micrographic evidence is presented to show that portions of the Golgi
are phagocytosed during spermiogenesis (see Fig. 18).
Subacrosomal Space
Expectedly, the subacrosomal space increased in volume as the acrosome spread over the nucleus. Its volume was maintained until step 19 (in stage VII), where
a decline in volume occurred. The decline has been
noted in a previous report and is probably due to escape
and phagocytosis of subacrosomal material via tubulobulbar complexes (Russell, 1979).
Nuage
Nuage in primary spermatocytes (all types; Russell
and Frank, 1978b) increased dramatically in pachytene
cells and dropped in volume as primary spermatocytes
divided to form secondary spermatocytes. Thereafter in
young spermatids, the only type of nuage present in
early spermiogenesis was the chromatoid body. The volume of the chromatoid body decreased markedly after
step 5 not to be present in step 15spermatids. Two other
forms of nuage were seen to appear at this time, the
granulated body and the reticulated body (Clermont et
al., 1990).Both increased in volume and then decreased.
The granulated body has been shown to label with antibodies directed against a variety of components of
outer dense fibers (Clermont et al., 1990), whereas the
reticulated body has shown no immunologic identity
with other spermatid components tested to date.
Other
The radial body attained its peak volume in step 14
spermatids and declined rapidly thereafter to not be
seen in step 19 spermatids. This organelle has a n elaborate structure (Clermont and Rambourg, 1978; Dym
and Cavicchia, 1978) and, to our knowledge, is not
found elsewhere in the body. Its formation and peak
203
GERM CELL MORPHOMETRY
development coincides with the development of the
outer dense fibers of the flagellum (Irons and Clermont, 1982) but, to date, no components of the outer
dense fibers have been associated with the radial body
in spite of attempts to do so (Hermo, personal communication).
The vesicular contents of spermatids posed classification problems to us in the present study since virtually none of the vesicular element have been described.
We classified them by simple descriptive terminology
without knowledge of function, if any, of any of the
vesicular types. Cytochemical information suggests
that one of these vesicle types, termed vesicles with a
granular interior, contain acid phosphatase activity
and may be part of the system of lysosomes (Bozzola
and Russell, 1992; Chemes, 1986).
Vesicles associated with dense material were seen a s
early as steps 9-11 of spermiogenesis. In following
them through spermiogenesis, the vesicles that appeared rounded originally appeared to flatten. Oko et
al. (1993) showed a micrograph of flattened vesicular
elements associated with h s e material in a step 18
that appeared similar to that described herein and suggested these may be part of the Golgi apparatus. More
work is needed to clarify the evolution of the vesicles
associated with dense material.
Chromatoid-associated vesicles show changes during
spermiogenesis. These changes can only be understood
by examination of both volume data and micrographs.
Initially (steps 1-4), the chromatoid-associated vesicles were small, but numerous. In steps 6 through 8,
they remained small but are less numerous contributing to a fall in the volume of such vesicles at these
stages. In steps 9-13 there were few vesicles, but all
vesicles were larger giving rise to increased volume of
vesicles per cell. Subsequently, the volume of chromatoid-associated vesicles fell in steps 14 and 15 as the
result of loss of vesicles. The functional significance of
such changes is not known.
The similarity of some vesicular elements with the
plasma membrane forming the flagellar canal may
lead one to confuse them in sectioned material. This
membrane of the flagellar canal is present from step 1
to step 15 0s spermiogenesis. At certain time periods in
spermiogenesis, vesicles and also the plasma membrane showed a n association with endoplasmic reticulum, creating the impression that both were of the
same membrane type (see Fig. 17). The similar properties of the membrane of these vesicles and the flagellar plasma membrane with respect to their ability to
maintain endoplasmic reticulum in close proximity
suggest that they might have a common origin.
Another structural element that has not been described, to date, is herein termed the discoid. We suspect it belongs to the family of lysosomes, although no
cytochemical evidence has been presented to that effect. Discoids reached their peak volume/per cell in
mid-pachytene at stage VI and declined thereafter.
This is the first study to analyze the volume and
surface area composition of the germ cell population in
any species. Virtually all other somatic cell types in the
testis have comparable data to that presented herein
for germinal cells, but the labor-intensiveness and considerable materials commitment necessary to complete
such a project have, to date, not encouraged such a
study. There are numerous published articles and reviews that describe germ cell ultrastructure, but the
contribution of the present study is that germ cell features are described quantitatively stage-by-stage, allowing investigators to follow their volume and surface
area parameters in association with the dynamic process of spermatogenesis. The data allow for the comparison of the relative amounts of structural components present at particular time periods that may give
clues to function andlor may shed light on biochemical
data obtained from specific cell types.
ACKNOWLEDGMENTS
The authors thank Dr. L. Hermo for helpful discussion about pertinent literature and for bringing to our
attention nuclear vacuoles in step 8 spermatids. The
help of Dr. J. Bozzola in regression analysis is also
greatly appreciated.
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