BIOLOGY OF REPRODUCTION 49, 1215-1228 (1993)
Surface and Surface-to-Volume Relationships of the Sertoli Cell during the Cycle of the
Seminiferous Epithelium in the Rat'
LUIZ RENATO DE FRANC(A, 4 SUSHMITA GHOSH, 3 SHI-JUN YE, 3 and LONNIE D. RUSSELL2 '3
Laboratory of StructuralBiology,3 Departmentof Physiology, Southern Illinois University, School of Medicine
Carbondale, Illinois 62901-6512
4
Institute
of Biological Sciences, Federal University of Minas Gerais
of
Morphology,
Department
Belo Horizonte, Minas Gerais, Brazil 31270-901 CP 2486
ABSTRACT
The surface relationships of the Sertoli cell and the surface relationships of the Sertoli cell in comparison to the changing
volumes of developing germ cells were studied using morphometric techniques at periods representing nine groupings of the
fourteen defined periods in the cycle of the seminiferous epithelium of the adult rat. No cyclic variation in the total Sertoli plasma
membrane surface area was noted. Cyclic variations were noted in the area of the Sertoli cell surface that faces the basal compartment germ cells, but not the basal lamina. No cyclic variations were noted in the amount of contact of the Sertoli cells with
each other at the level of the Sertoli cell barrier. However, when areas in the adluminal compartment were studied, significantly
less Sertoli-Sertoli contact was seen in stages V through VII than in other stages with the exception of stages II-IV. Surface
contact of germ cells with Sertoli cells increased progressively as germ cells entered the intermediate compartment and progressed to late spermatids. However, a calculation of the surface-to-volume ratio showed that surface increases of the Sertoli cell
in relation to the volume of germ cells were greatest in elongating spermatids past step 12 of spermiogenesis. The area in which
Sertoli ectoplasmic specializations faced germ cells was determined throughout spermatogenesis, and these data demonstrated
that the first appearance of ectoplasmic specialization was at the mid-pachytene phase. They also showed that stage VIII was a
period when ectoplasmic specialization loss from the cell surface was evident. Less Sertoli ectoplasmic specialization faced step
8 and step 19 spermatids than comparable germ cell types at other stages. In addition to Sertoli cell surface area changes during
the cycle, volumes of individual germ cell types were determined for the first time. The data presented allow an objective
understanding of the complex structure and relationships of the Sertoli cell and provide a basis for understanding functional
changes and interpreting biochemical data.
INTRODUCTION
Since the Sertoli cell is thought to be an important, if
not the most important, mediator of hormone action [1, 2]
with respect to support of the germinal cell population of
the testis, it is useful to know how the Sertoli cell interacts
with neighboring germinal elements. Interactions can be
biochemical [3, 4] and/or structural [5, 6]. In general, functional changes are accompanied by morphological manifestations of such changes.
The organization of the seminiferous epithelium in
mammals demonstrates that spermatogenesis is cyclic. The
kind and extent of structural relationships of the Sertoli cell
to other cell types and acellular elements certainly play an
important role in cyclic functional events. Moreover,
knowledge of Sertoli-germ cell relationships in itself helps
one understand and envision this complex epithelium and
the modifications that both germ cells and Sertoli cells must
undergo during the cyclic events of spermatogenesis. The
most extensive study to date of surface area relationships
of the Sertoli cell was undertaken in a reconstruction of a
model of rat and monkey Sertoli cells [5, 7, 8]. However, the
Accepted July 27, 1993.
Received May 6, 1993.
'This work was supported in part by a fellowship awarded to Luiz Renato de
Franca from the Brazilian Research Council (CNPq).
2
Correspondence. FAX: (618) 453-1517.
1215
data from that study represented only one cell from each
of two different species. In those experiments it was not
possible to determine cyclic variations in Sertoli cell relationships. Thus the objective of the present study was to
determine the cyclic variation in Sertoli cell surface relationships in one species, the rat.
MATERIALS AND METHODS
The present study employed micrographs taken to analyze the volume composition of the subcellular components of the seminiferous epithelium. In brief, the testes of
four adult male rats were perfused-fixed with glutaraldehyde and the tissue was embedded in Araldite. Thin sections were made for electron microscopy of tubules at selected stages of the cycle. Pie-shaped [9] montages made
from negatives taken at 2400 times and enlarged to 5900
times original size of round or near-round tubular profiles
were constructed from individual electron micrographs to
provide proportional sampling of the seminiferous epithelium [10].
Morphometry for surface area measurements was conducted using a Mertz grid overlay on the pie-shaped montages according to standard stereological methods [11]. To
make surface area determinations, points over the Sertoli
cell as well as intersections with the Sertoli cell plasma
membrane were counted and recorded.
1216
FRAN(A ET AL.
2
TABLE 1. Surface parameters (m ) of the Sertoli cell (mean - SE).
STAGES
Plasma membrane
I
Il-IV
V
VI
VII
VIII
IX-XI
XII-XIII
XIV
14,348 ± 1,592
15,331 + 670
15,804 ± 1,993
15,567 + 1,130
13,960 ± 1,142
12,472 ± 1,621
11,913 + 1,104
13,147 ± 1,622
12,842 ± 1,214
Sertoli cell contact with:
527 +
369 ±
25
566 + 33
381+ 32
878 ±
416 ±
58
59
483 +
397 +
26
77
387 +
358 +
48
74
972 ±
423 +
47
31
914 ±
454 ±
54
73
32
550 ± 40
65
389
523 +
381 ±
77
16
158 ±
58
185 ± 11
462 ±
86
460 ±
76
549 ±
82
29 +
29
86 ±
14
161 + 47
142 ±
70
with ectoplasmic specialization
871 ±
295
740 + 107
823 ±
176
954 ±
133
684 ±
174
452 ±
45
838 ±
157
578 ±
165
617 +
71
without ectoplasmic
specialization
597 ±
194
462 ± 99
474 ±
115
506 ±
91
435 ± 78
352 +
41
476 +
102
310 ±
98
469 ±
66
274 ±
103
278 ± 17
349 ±
87
448 +
59
249 +
100 +
33
362 ±
59
268 +
67
148 ±
52
744 ±
109
Basal compartment
Basal lamina
Basal compartment germ
cells
Sertoli barrier
Intermediate compartment
126
511 ±+ 100
(primary spermatocytes)
Adluminal compartment
Primary spermatocytes
Primary/secondary sperma-
12,950 ± 1,317
752 ±
162
14,025 ± 585
1,205
14,103 ± 1,816
13,699 ± 1,016
12,304 ± 1,102
11,122 ± 1,494
9,848 ± 1,126
12,019 + 1,585
45
202
11,702 ± 1,153
792 ±+ 101
2,798 ± 277
119
1,566 +
42
1,530 ±
217
2,024 ±
283
1,953 +
150
2,027 +
212
689 +
3,279 ±
686
681
5,248 ±
947
5,877 ± 1,090
5,728
480
2,104 ±+ 204
469 ± 164
1,735 ±+ 343
439 ± 48
2,139
245
496
69
1,518 ±
114
tocytes
Round/elongate spermatids
Elongate spermatids
3,475
3,810 + 184
4,742 +
5,369 ±
607
4,982 ±
444
4,376 ±
6,441 ±
720
7,785 ± 385
6,868 ± 1,029
5,696 +
685
3,053 ±
507
Sertoli cells
Tubular lumen
2,135 ±
147 ±
408
32
1,081 ± 105
144 + 77
815 ±
112 ±
298
33
864 ±
240 +
231
79
784 ± 234
1,461 ± 404
1,515 ± 622
2,102 ± 567
1,176 + 303
1,464 ±
272
1,192 ± 98
1,347 ±
214
1,290 ±
237
1,019 +
55
655 ±
103
624 ±
840 ±
215
121
473 ±
874 ±
115
173
514 ±
776±
90
199
435 ±
78
352 ±
584±
65
303 ±
± 492
Sertoli plasma membrane associated with ectoplasmic specialization
Sertoli-Sertoli
1,236 ±
51
1,184 -
126
41
476 ±
102
316 +
98
139
708
82
920±
136
(throughout)
(epithelium)
Germ cells
Primary spermatocytes
Primary/secondary
spermatocytes
Round/elongate
spermatids
Elongate spermatids
482 ± 105
710 + 87
0
0
0
840 ±
121
133 37
577 ± 91
204 ± 41
670 + 139
76 ±
700 ±
The Sertoli cell plasma membrane was divided into sections based upon the cell or structure that opposed that
particular portion of the cell. The cell surface was first divided into major categories: surfaces forming the basal, intermediate, and adluminal compartments [12-14]. These
surfaces were then subdivided into regions that comprised
the body and cell processes. The body and cell processes
were defined according to Wong and Russell [7]. Sub-subdivisions of the cell were considered with regard to the
particular acellular or cellular element facing the Sertoli
cell. These included the basal lamina, basal compartment
germ cells, other Sertoli cells including the region forming
the Sertoli cell barrier, primary spermatocytes, secondary
spermatocytes, round spermatids, elongate spermatids, residual bodies, and free surfaces facing the lumen. These
sub-subdivisions were again subdivided into categories depending on whether or not the particular region of the Sertoll cell contained ectoplasmic specialization on its surface.
Table 1 shows the subdivisions used to record surface relationships of the Sertoli cell.
The micrographs utilized did not encompass the central
lumen; thus quantitative measurements did not include a
6 ±
0
0
0
26
215
13 ±
571 ±
8
67
293 ±
4 ±
82
140
0
0
0
0
501 ±
1,017 ±
4
137 ±
41
70
11
137
4
571 ±
45
850
126
64
953±
35
142
portion of the flagellum in the lumen (the principal and
end pieces; see Discussion).
The surface density (Sv) of particular subsections of the
plasma membrane was determined by the following formula:
2xi
Sv =
Pt x Z
where I is the number of intersections on the Sertoli cell
plasma membrane, Pt is the number of points over the Sertoli cell, and Z is the length of the line between points in
terms of the magnification of the micrograph.
To determine surface (S) area from surface density, the
following formula was applied:
S = Sv x V,
where V equals the volume of the Sertoli cell. The figure
used for the Sertoli cell volume was that determined by Ye
et al. [15]. This figure represents the mean volume of all
Sertoli cells during the cycle of the seminiferous epithelium in the same animals. The mean volume was utilized
1217
SURFACE OF THE SERTOLI CELL DURING THE SERTOLI CELL CYCLE
Plasma
Sertoli Cell Contact with Basal
Compartment Germ Cells
Membrane
18000
700 a
16000
yiik
14000
CaM
E
600 -
12000
c4
a
a
500-
E
10000
400-
8000
E
0
U
300-
6000
0
4000
0
2000
100-
0
I
II-IV
V
VI
VII
VIII IX-XI XII-XIII XIV
Stage of cycle
I
V
I
II-IV
V
FIG. 1. Sertoli cell surface area. No statistical differences were noted.
since it was found that no significant cyclic variation in cell
volume existed.
The volume of a particular germ cell population associated with a single Sertoli cell was determined by the following formula:
mean volume of a single Sertoli cell
mean volume of particular germ cell population
associated with a single Sertoli cell
Vv Sertoli cell in epithelium
Vv particular germ cell in epithelium
This figure, i.e., the mean volume of a population of a particular germ cell type within the epithelium at a particular
Sertoli Cell
with Basal
600
VI
Stage
VII
VIII
IX-XIXII-XIII
XIV
of cycle
FIG. 3. Sertoli cell contact with basal compartment germ cells. Data
points with like lettering are not different from each other, whereas data
points with dissimilar lettering represent significant differences.
stage, was divided by the number of germ cells of a particular type known to be associated with a single Sertoli cell
(data from [16]).
Means from each animal were analyzed by ANOVA and
a significance level of p < 0.05 was used to determine a
significant difference. The Student-Newman-Keuls test was
used to determine which groups were different.
RESULTS
Surface areas are presented in tabular (Table 1) and
graphic form (Figs. 1-16). The table provides the values
Contact
Lamina
Sertoli Cell Contact with Basal Compartment (BC)
and Forming the Adluminal Compartment (AC)
16000 -
-
14000.
500 -
12000 -
N'
E
Co
400 -
E
a
300 -
0
a
10000 -
8000 BC-
6000-
W
AC
4000 -
200
2000-
Co
100
b
0
I
.
I
. . .... -. . . *. . m*-. ..m . . . I. . .
II-IV
V
VI
VII
Vil IX-XIXII-XIII XIV
Stage of cycle
b
ba
b
a
Il-IV
V
b
a
bI
.
b
b
.
VI
VI
VIII IX-XI XII-XIII XI
Stage of cycle
· m
FIG. 2. Sertoli cell contact with the basal lamina. No statistical differences were noted.
FIG. 4. Sertoli cell contact forming the basal compartment and adluminal compartment. Data points with like lettering are not different from
each other, whereas data points with dissimilar lettering represent significant differences. No statistical differences were noted in the adluminal
compartment.
1218
FRANCA ET AL.
Sertoli Cell Surface Area Forming the
Region of the Sertoli Cell Barrier
Sertoli Cell Surface Displaying
Ectoplasmic Specialization
1200900-
800
1000 -
700E
800 -
C1
E
600 -
a
E
400 t
n8
200 -
I
II _
I-V
; 'I-I'
V
VI
I
XX
VI
400300-
=
200100-
V' ' V V' Vil IX-XIXII-XIII, XIV
Stage
500-
u
I
I-II
600-
[I
of cycle
v
i
.
I
.
.
Il-IV
.
.
V
(A)
Sertoli-Sertoli Contact at Sites
other than the Sertoli Cell Barrier
b
.
.
.
.
.
.
.
.
B
FIG. 6. Sertoli-Sertoli contact showing ectoplasmic specialization
throughout the entire seminiferous epithelium. No statistical differences were
noted.
Sertoli Cell-Basal Compartment Contacts
2000 -
E
a.
a 1500-
£
a 100019
U
4n
a
500 -
n
.
b
2500 01
.
VI
VII
Vill IX-XI XII-XIII XIV
Stage of cycle
.
.
I
II-IV
V
.
..
..
.
.
VI
VII
VIII IX-XI XII-XIII XIV
Stage of cycle
(B)
The surface area of the Sertoli cell that makes contact
with the basal lamina did not show a significant cyclic change
(Fig. 2), although a slight trend showing a peak at stage VI
is indicated from the pattern of the graph (see Discussion).
Surface contact with basal compartment germ cells shows
a significant cyclic variation (Fig. 3). The Sertoli cell in stages
V through VII shows a significantly greater surface area facing basal compartment germ cells than in all other stages.
The surface relationships of the Sertoli cell as it forms the
basal compartment (contact with basal lamina and basal
compartment germ cells) reflects the same significances
(Fig. 4) described above for surface relationships with basal
compartment germ cells alone.
FIG. 5. Sertoli-Sertoli contact. A) Sertoli-Sertoli cell contact in the region of the Sertoli cell barrier. No statistical differences were noted. B) Sertoli cell contact with other Sertoli cells in regions other than those forming
the Sertoli cell barrier. Data points with like lettering are not different from
each other, whereas data points with dissimilar lettering represent significant differences.
Total
Sertoli-Sertoli
Contact
361
321
obtained and the figures are provided as a visual display of
the pattern of cyclic changes within the seminiferous epithelium. Statistical results from Table 1 are presented in the
text on key comparisons. Statistical comparisons are provided in the figures.
Nc
281
E
24(
20C
164
t
Cell Surface Area
The Sertoli cell plasma membrane surface area is shown
in Figure 1. There were no significant cyclic differences,
although a trend was noted that suggested a decrease in
surface area at mid- and late cycle (stages VIII through XIV).
a4
8
4'
I
II-IV
V
VI
VII
VIII
IX-XI XII-XIII
XIV
Stage of cycle
FIG. 7. The total surface area of the Sertoli cell showing contact with
other Sertoli cells. No statistical differences were noted.
1219
SURFACE OF THE SERTOLI CELL DURING THE SERTOLI CELL CYCLE
Cell
Sertoll
Contact
Sertoli Cell Contact with
the Tubular Lumen
with Spermatocytes
3500 -
2000'
3000 -
1750
2500 E
CNI
2000-
E
b
·
1500
1250'
1500-
1000'
4
1000 rb
e
500 -
750'
500
VIIl
Pi
IX-XI XII-XII XIV
L
Z
P
P
P
II-IV
P
V
V
P
P
P
Stage of cycle
VII
P
Vil IX'XI XII-'XIII XIV
P
P P/Di 2nd
a
·
a
250'
I
(A)
4000-
*
3000-
zzH''
1000-
I
'
II-IV
VI ' V
V
VIII
Stage of cycle
(B)
9000
Sertoll Cell Contact with
Elongating Spermatids
8000.
-
7000
E
6000.
VI
VII
IX'XI XII-XIII
XV
cycle) of the barrier was shown to be lined by ectoplasmic
specialization. When the components of the barrier were
analyzed separately, it was noted that neither the ectoplasmic specialization-associated nor the non-ectoplasmic
specialization-associated regions of the cell showed a significant cyclic variation (graphs not shown).
The surface area where Sertoli cells contact each other
in regions other than those forming the Sertoli cell barrier
is shown in Figure 5B. These regions were all formed by
the adluminal compartment surface of the Sertoli cell. Stages
V through VII show significantly less Sertoli-Sertoli contact
than all other stages except stages II-IV.
Figure 6 depicts the surface area of the Sertoli-Sertoli
cell contact throughout the entire epithelium showing ec-
2000
0
VI
FIG. 9. Sertoli cell contact with the tubular lumen. Data points with
like lettering are not different from each other, whereas data points with
dissimilar lettering represent significant differences.
6000-
E
II-IV 'V
Stage of cycle
Sertoll Cell Contact with
Round Spermatlds
5000-
a
ac
5000,
a
Sertoli Cell Contact (Showing Ectoplasmic
Specialization) with Germ Cells
3000.
1200-
2000
1000-
i
IX-XIXII-XIII XIV
-
X
.
.
.
I
II-IV
V
Stage of cycle
.
1000 -
.
VI
VII
VIII
(C)
FIG. 8. Sertoli cell contact with (A) spermatocytes, (B) round spermatids, and (C)elongate spermatids. Data points with like lettering are not
different from each other, whereas data points with dissimilar lettering represent significant differences. No statistical comparisons were made in (A).
cI
E 800 6004
0
co
400-
0
200-
Sertoli-Sertoli Contact
The surface area in which individual Sertoli cells make
contact with each other in the region of the Sertoli cell
barrier showed no significant cyclic variations (Fig. 5A). When
expressed as a percentage of the total area of the Sertoli
cell barrier, approximately 65% (mean value during the
n
.
.
I
.
.
Il-IV
.
.
V
.
VI
VII
VIII
Stage of cycle
IX-XI XII-XIII
XIV
FIG. 10. The Sertoli cell surface displaying ectoplasmic specialization
facing germ cells. Data points with like lettering are not different from each
other, whereas data points with dissimilar lettering represent significant
differences.
1220
FRAN§A ET AL.
a
D,
o0n
mt
C
E
:
CL
LU
c
-o
:0
C
C-
E
_
o
L
o C
'
EE
ci,
E
a
C
c,-
_
U
-
w
r
E
_ Nw
4
·i~
0
C
C
o
0
co
0
E
Cc
0
a
u
.2
.
C
2
-a
a
V-
C
o
0
ci,
E
C)
a
:
0)
C)
Cl
o
sF-
60~~~~~~(
LLz
o
§
oe° 'l n ee
jo S
wurl)
eeny eospnS
8
°
m
E
SURFACE OF THE SERTOLI CELL DURING THE SERTOLI CELL CYCLE
Surface Area of the Body
of the Sertoli Cell
70
1221
(Fig. 8A). None of the surface area of the Sertoli cell facing
intermediate compartment germ cells displayed ectoplasmic specialization (Table 1).
The area of the Sertoli cell that faces each specific germ
cell type is shown in Figure 8.
b
60
50
a
Sertoli Cell-Adluminal Compartment Contacts
40
30
20
10
0
-
s
b
·
.
II-IV
V
,
.
*
VI
Vil
VIII
. -
IX-XIXII-XIII
XIV
Stage of cycle
(A)
Surface
Area of the Processes
of the Sertoli Cell
a
7060 -
50 40 30 20 -
10-
I
iI-IV
V
Vi
VII
VIII IX-XI XI-XIII
Stage of cycle
XIV
(B)
FIG. 12. The Sertoli cell surface forming the body (A) and cell processes (B). Data points with like lettering are not different from each other,
whereas data points with dissimilar lettering represent significant differences.
toplasmic specialization. No significant cyclic differences were
noted. The total surface area displaying Sertoli-Sertoli contact is shown in Figure 7. Stages II-VII showed a trend,
although not significant, for lower Sertoli-Sertoli contact than
did other stages.
Sertoli Cell-Intermediate Compartment Contacts
When the intermediate compartment is defined as the
relationship of Sertoli cells with preleptotene-leptotene
transition cells and with leptotene cells in stages VIII-XI,
there is a progressive increase in the surface area where
the Sertoli cell faces stage IX-XI leptotene cells as compared with stage VIII preleptotene-leptotene transition cells
The total adluminal compartment surface relationships
made by the Sertoli cell are shown in Figure 4. Although
no significant differences were noted, a trend toward decreasing adluminal compartment surface area was noted
beginning at stage VII and continuing to stage XI.
The surface area of individual Sertoli cells facing primary and secondary spermatocytes is depicted in Figure 8A.
There was a general trend for the Sertoli cell surface area
that faced spermatocytes to increase as these cells matured.
There was no difference in the surface area contact of Sertoli cells with secondary spermatocytes as compared with
late pachytene and diplotene spermatocytes, nor was there
a significant change in the contact of Sertoli cells with secondary spermatocytes compared with step 1 spermatids (see
Fig. 8, A and B).
The surface area where the Sertoli cell faced round spermatids (considered to be in stages I through VIII) did not
show significant differences (Fig. 8B). There was a trend
for Sertoli cells to increase their contact with round spermatids until step VI and to decrease in steps VII-VIII.
The surface contact of Sertoli cells with elongate spermatids (Fig. 8C) showed a trend toward increasing in steps
9 through 16 and a decline beginning at step 17; this value
becomes significantly lower at step 19 than those for all
other elongate spermatids.
The surface relationship of the Sertoli cell with the tubular lumen (Fig. 9) increased significantly in stages VII and
VIII compared to all other stages.
Sertoli Cell Ectoplasmic Specialization in Contact with
Adluminal Germ Cells
Data for the surface area of the Sertoli cell containing
ectoplasmic specialization are provided in Table 1. Significantly less ectoplasmic specialization was noted in stage
VIII than in stages I and XIV.
Figure 10 represents the surface of the Sertoli cell showing ectoplasmic specialization facing germ cells. Stage VIII
shows less ectoplasmic specialization facing germ cells than
stage XIV.
Figure 11 shows that ectoplasmic specialization of the
Sertoli cell begins to face the population of germ cells at
the pachytene phase of meiosis in stage VIII. The surface
area showing ectoplasmic specialization remains low until
a progressive increase is seen in relation to step 8 spermatids and all subsequent elongating spermatids except at
step 19. In stage VIII, the surface area of ectoplasmic specialization facing step 19 spermatids decreased dramatically
1222
FRANCA ET AL.
TABLE 2. Volumes (m3 ) of individual germ cells (mean + SE).
Stages
Primary spermatocytes Primary/secondary spermatocytes Round/elongate spermatids Elongate spermatids
1
II-IV
V
VI
VII
VIII
IX-XI
XII-XIII
XIV
658 ± 135
438
62
413
129
543
91
497 +±40
756 - 58
1112
1541
1777
2498
2816
2624
4202
2507
±
±
±
+
+
+
+
+
149
134
172
118
223
225
123
220
compared to that of previous elongating/elongate spermatids.
Expressed on a per cell basis, the area of ectoplasmic
specialization facing each germ cell type is similar to that
shown in Figure 11 (data not shown).
Surface Areas of the Sertoli Cell Bordering the Body or
the Processes of the Cell
The surface area of the Sertoli cell that is manifested in
the body of the cell and in cell processes (see Materials
and Methods for definition of cell body and cell processes)
is shown in Figure 12. Approximately 43% of the total surface of the Sertoli cell (mean value during the cycle) forms
the body of the cell and approximately 57% forms the cell
processes. The body of the cell contains significantly more
surface area in stages VII than in stages II-IV. Significances
are similar but opposite results are noted when processes
of the cell are considered.
Volumes of Individual Germ Cell Types and Surface-toVolume Relationships
The volume of individual germ cells (Table 2) was calculated as described in the Materials and Methods section
and the data are shown in Figure 13A. There is an expansion of basal compartment germ cells in stages I through
VII followed by a sharp drop in their volume in stage VIII.
Slow growth of the volume of basal compartment germ cells
is suggested from stage VIII to XIV.
The extent of surface area relationship of the Sertoli cell
with individual germ cell types is shown in Figure 13B and
plotted along with the volumes of individual germ cell types.
Surface-to-volume relationships (i.e., the surface area of a
Sertoli cell expressed as a ratio to the volume of adjoining
germ cells) are shown in Figure 14. All preleptotene, leptotene, and pachytene spermatocytes and spermatids up to
step 9-11 show a relatively low surface-to-volume ratio with
the Sertoli cell. Beginning with step 12 spermatids, the area
of the surface where the Sertoli cell faces the germ cell
increases in porportion to the volume of the germ cell and
is maintained throughout the completion of spermiogenesis.
A summary figure showing the relative surface area of
the Sertoli cells in relation to cellular and acellular com-
1279 - 87
1170 - 94
1439
1682
1890
1900
1493
1254
1139
1173
1239
1090
876
477
242
+-39
+ 171
+ 141
+ 72
± 116
+ 25
+ 138
+ 94
± 76
+ 175
± 79
+ 95
± 91
ponents in the seminiferous epithelium is provided
(Fig. 15), as is a summary figure showing the volumes of
cells composing the epithelium (Fig. 16). These figures are
provided to visually enhance understanding of the surface
relationships and cell volumes as a function of the spermatogenic cycle. The top of Figure 16 reflects the total cumulative volume of the seminiferous epithelium. Statistical
analysis (not shown in Fig. 16) indicates that for stage IXXI the volume of the epithelium is significantly lower than
for stages II-VII.
DISCUSSION
Less is known about the surface relationships of the Sertoli cell than about any other parameter of this cell. Most
information presented to date is on the total plasma membrane surface area (summarized by [15]) or gives a breakdown of the Sertoli cell surface area during one particular
stage of the cycle in one cell [5, 8]. Although the data presented here are descriptive, they allow a quantitative (objective) understanding of how the Sertoli cell relates to other
components of the tubule without reliance on the subjective interpretations of electron micrographs. Moreover, they
allow a comparison of cyclic differences in the Sertoli cell
in relation to the production of sperm. In the process of
determining the above, it was possible to also determine
the volume of individual germ cell types, something that
has not been presented in the past.
Cell Surface Area
Although examined by a separate investigator, the surface area of the Sertoli cell as reported here is comparable
in amount and magnitude to that determined in a recent
study from this laboratory [15] and similar to that determined by reconstruction of a stage V rat Sertoli cell [17].
Although no significant cyclic differences were recorded,
there is a trend for the surface area to be decreased beginning at mid-cycle (VII) and continuing throughout most
of the latter portion of the cycle. This was also noted in
another study [15] suggesting that surface area may be decreased as a result of endocytotic uptake occurring in this
period [18-20] or as a result of decreasing membrane surface area due to a fundamental change in the relationship
of Sertoli cells to germ cells at spermiation, or both.
1223
SURFACE OF THE SERTOLI CELL DURING THE SERTOLI CELL CYCLE
Sertoli Cell-Basal Compartment Contacts
and Christensen's data [16], there is a sharp drop in the
tubular diameter in stage IX, just as spermatocytes move
upward, that compensates for the loss in contact of the germ
cells with the basal lamina. Overall, these data support the
There were no cyclic differences in the absolute area of
contact of the Sertoli cell with the basal lamina, although
a minor (not significant) trend towards an increase in contact was noted in stage VI. This result would not have been
expected, since the basal compartment germ cell population was the largest in terms of numbers of cells. One explanation for the fact that the data show no change in basal
contact of the Sertoli cell, in spite of an expanding population of germ cells, is a possible compensatory increase in
the tubular diameter that occurs in parallel with the expansion of the basal compartment germ cells in stages IV
through VIII [16]. Since tubules are known to be larger at
certain stages [16], basal lamina may be stretched cyclically
in response to germ cell proliferation. It is also possible
that individual germ cells as they divide along the basal
lamina grow little and/or lose their flattened character along
the basal lamina and take up progressively less and less
basal lamina space.
The data also suggest that the contact of the Sertoli cell
with the basal lamina is altered minimally as spermatocytes
make their upward migration [13]. But as noted from Wing
Basal
Compartment
Germ
Cells
12001000-
800-
C
E
6000
E
·
400200-
I]
v
-
-
I
-
II-IV
-
-
- -. -.
V
VI
.-
..
vI
VIII
IX-XI XII-XIII XIV
Stage of cycle
(A)
Relationship of the Surface Area of Sertoll
Cells to the Volume of Germ Cells
4500
4000
Volume
35o
o
E
3000
e2
o
2500
2
1500'
(a
1000
Surface Area
5soo00
0
-
.
Vill
.
.
.
IX-XI XII-'XIII
Xiv
.
.
I
.
.
.
.
l-.IV V
.
.
VI
.
.
Vll
.
.
.
.
.
-
-
-
Vill IX-XI XII-XIII XIV
Primary spermatocytes
-
I
-
-
- --
II-IV V
-
V;
-
vII
Round spermatids
-·
VIII IX-XI'XII-XIII xIV
I
II-IV
V
v
VI
II
VII
Elongating/elongate
cnnrmatiis
(B)
Stage
of cycle
FIG. 13. (A) Volumes of germ cell in the basal compartment. (B) The Sertoli cell surface that faces individual germ cell types and the volumes of
individual germ cells. No statistical analysis was performed for (B).
1224
FRANQA ET AL.
SURFACENOLUME
h_ 1.0
0.9.
0.8s
0.7'
0.6-
0.5'
0.4
0.3-
0.2
0.1-
V;ll
IX-XIXII-XIII XV
I
I-IV
V
VI
VII
VIII
IX-XIXII-XIII XIV
I
II-IV
V
VI
VII
Round spermatids
Primary spermatocytes
VIII
IX-XIXII-XIII XIV
I I-IV
V
VI
VII
VIII
Elongating/elongate
spermatids
Stage of cycle
FIG. 14. The Sertoli cell surface facing individual germ cell types expressed as a ratio to the volumes of individual germ cells. No statistical analysis
was performed.
concept that the Sertoli cell is cyclically static with respect
to its relationship to the basal lamina but that the basal lamina itself undergoes cyclic fluctuations in surface area. There
must be some cyclic fluctuation in coverage of the basal
lamina by the Sertoli cell, since at stages VIII and IX there
is migration of Sertoli cell processes to undermine and assist in upward movement of spermatocytes [13]. This movement must increase the cell surface exposed to the basal
lamina, at least transiently until division of A spermatogonia
occurs.
The data on surface area contact of the Sertoli cell with
the basal compartment suggest that germ cell proliferation
is responsible for significant increases in the size of the
basal compartment (compare Fig. 4 with Fig. 13A). Proliferation of germ cells in stages II through VII dramatically
increases the area of Sertoli cells exposed to these germ
cells. Movement of germ cells into the intermediate compartment of the testis, on the other hand, results in a significant drop in these surface area relationships. Thus the
overall pattern of the basal contact of the Sertoli cell
(Fig. 3) is more a reflection of contact with germ cells than
of contact of the Sertoli cell with the basal lamina.
Sertoli-Sertoli Contact
Sertoli-Sertoli contact in the region of the Sertoli cell
barrier was not significantly cyclically altered. There was,
however, a trend indicating that the area of Sertoli-Sertoli
contact was depressed in stage VIII. This decrease is due
FIG. 15. A summary figure showing the surface relationships of the
Sertoli cell during the spermatogenic cycle.
SURFACE OF THE SERTOLI CELL DURING THE SERTOLI CELL CYCLE
1225
I
Lumen
A
D
L
U
M
I
N
Late Elongating
Spermaticis
1
Comptt
l
II-IV
V
VI
VII
STAGE
VIII
IX-XI
XII-XIII
XIV
1226
FRANCA ET AL.
ET- A ..
... . - ---
- 35,000
- 30,000
- 25,000
- 20,000
0
rr
l
15,000
3
10,000
- 5,000
-_
I
II-IV
V
VI
VII
VIII
IX-XI
XII-XIII
XIV
STAGE
FIG. 16. A summary figure showing the volumetric composition of the seminiferous epithelium during the spermatogenic cycle.
to loss of non-ectoplasmic specialization regions rather than
ectoplasmic specialization-containing regions of the cell. The
non-ectoplasmic regions of the barrier in this stage are possibly affected by the movement of the primary spermatocytes from the basal compartment to the intermediate compartment [13,14].
The percentages of the cell surface at the Sertoli cell
barrier with and without lining ectoplasmic specialization
are approximately similar to those given in the study by
Weber et al. [17] showing that about two-thirds (mean of
the cycle) of the Sertoli-Sertoli contact in the general region of the barrier is lined by ectoplasmic specialization.
With respect to the possibility that this is a reflection of the
Sertoli-Sertoli junctions being more extensive at particular
stages and thus occupying more area, there is no significant
difference in the area of ectoplasmic specialization that would
indicate that one stage shows a tighter epithelium than another.
The area of the cell surface in regions other than those
forming the Sertoli cell barrier does show interesting cyclic
variation in Sertoli-Sertoli contact, with stages II-VII showing significantly less contact. The reason for this is not known.
Sertoli-Germ Cell Contact
The area of the cell surface at which the Sertoli cell contacts the germ cells is generally a reflection of germ cell
growth. When the data for Sertoli cell surface area contact
are plotted against cell growth, the lines are generally parallel (Fig. 13B). Some findings are worthy of comment. It
seems that after cell division in meiosis, the Sertoli cell does
not immediately re-establish extensive surface contact with
the secondary spermatocytes and that this is to some degree the case with step 1 through 8 spermatids, which during the process of division must have themselves, as a cell
population, increased in surface area.
SURFACE OF THE SERTOLI CELL DURING THE SERTOLI CELL CYCLE
1227
Surface-to-volume (Fig. 14) relationships indicate that the
Sertoli cell establishes proportionally more contact with
elongating germ cells than with other cell types. It does so
by placing germ cells in crypts of the Sertoli cell [21] and
by forming penetrating processes [22, 23]. The increases in
the surface-to-volume relationship possibly reflect a need
for Sertoli-germ cell transfer of metabolites. The relative
increase in this relationship appears to be unique to elongating spermatids, although the specific substances transferred from one cell to another are not yet known. Perhaps
elongate spermatids are less metabolically active than other
germ cell types and are more dependent on Sertoli cells.
A significantly greater surface area of the Sertoli cell faces
the tubular lumen in stages VII and VIII than at other stages,
a result that could be anticipated on the basis of the formation of the apical cytoplasmic lobe that extends the late
spermatids into the tubular lumen [24, 25].
Surface Areas of the Sertoli Cell Bordering the Body or
the Processes of the Cell
Sertoli Cell Ectoplasmic Specialization in Contact with
Adluminal Germ Cells
Volumes of Individual Germ Cell Types and Surface-toVolume Relationships
The present investigation agrees with most previous
studies showing the distribution of ectoplasmic specialization along germ cell types. No ectoplasmic specialization is
seen facing germ cells prior to the development of midcycle pachytene spermatocytes. At stage VIII, a small amount
of ectoplasmic specialization faces pachytene cells and newly
elongating spermatids. We believe that our finding that no
ectoplasmic specialization faced step 1 spermatids does not
reflect what occurs normally. Examination of tubules other
than the ones used showed ectoplasmic specializations of
the Sertoli cell facing step 1 spermatids. At step 7 of spermiogenesis there is a decrease in ectoplasmic specialization that we believe may be a real occurrence. This decrease in ectoplasmic specialization parallels the decrease
of ectoplasmic specialization seen along step 19 spermatids
(this study; [21, 25, 26]), which has been suggested to occur
as a result of depolymeration of actin. (Ectoplasmic specialization that was internalized from step 19 spermatids
was not quantified in the present study since it was not a
surface structure.) Thus it may be a general phenomenon
within the Sertoli cell to depolymerize actin at stage VII.
The surface area of ectoplasmic specialization of elongate spermatids progressively increases in spermatids
undergoing elongation (steps 8 through 14), probably as a
result of recycling of some ectoplasmic specialization from
step 19 spermatids but also because of recruitment of new
ectoplasmic specialization from an actin pool [25,26]. A
precipitous drop of ectoplasmic specialization in step 19
spermatids has been indicated from previous studies suggesting that the tubulobulbar complexes are involved in receptor-mediated endocytosis of junctional links between the
germ cell and Sertoli cell that causes the release of ectoplasmic specialization from the surface of the Sertoli cell
and its movement into the cytoplasm [25, 27].
In the course of determining surface relationships we
were able to determine the volumes of individual germ cells
on the basis of our data and data from Wing and Christensen [16]. To our knowledge, such data for all germ cell types
have not been available, although limited data on spermatocytes have been presented [28]. A knowledge of the
volumes of germ cells is fundamental to understanding the
process of cell growth, synthetic processes, and cytoplasmic
elimination during spermiogenesis.
From the growth curve presented, preleptotene, leptotene, zygotene, and early pachytene phases of spermatocyte
development are slow-growth phases. Rapid spermatocyte
growth begins in stages II-IV of the pachytene and continues through the diplotene phase. Slight cell growth appears
to take place in secondary spermatocytes and step 1 spermatids, since the values recorded are higher than would be
expected by simple halving of cytoplasmic volumes due to
cytokinesis.
Growth of round spermatids in stages I through VII is
indicated, although no significant differences were determined. However, at stage VIII, the cell size of step 8 spermatids shows a decreasing trend until step 12, where cell
size is relatively stable until step 17. Loss of cell volume in
newly elongating cells may be due to a decrease in nuclear
volume during chromatin condensation. From step 17 there
is a progressive decrease in cell size until spermiation. Decreased cell size in elongated spermatids, first indicated by
Roosen-Runge [29], has been suggested to be caused by loss
of water from the cell [30] as well as by elimination of cytoplasm due to degradation of tubulobulbar complexes
[18,31]. The size of the elongate spermatids has been
underestimated slightly in the present study since the principal pieces of the flagella were not counted as part of the
cell volume.
Division of the body of the cell from its cell process on
the basis of criteria provided by Wong and Russell [7] shows
that stage VII is substantially different from stages II-IV in
that it contains significantly more cell processes. This was
the basis for originally [7] dividing the Sertoli cell into two
configurations (A and B) that would reflect the two major
cyclic configurations of the cell. Type A Sertoli cells in the
rat contain more processes since these processes form the
crypts for elongating spermatids. Type B Sertoli cells show
an elongate body that reaches the lumen. These definitions
are useful in that they aid in visualization of the cyclic changes
of the cell. It remains to be determined to what extent the
cell changes its cytoskeletal components to make these two
configurations of cells.
1228
FRANCA ET AL.
Volume Density of the Sertoli Cell
In the course of making the calculations presented herein,
it was necessary to determine the volume density of the
epithelium as a function of the cycle. A previous study [15]
did not show significant cyclic variations in volume density,
although the same general pattern was noted as seen in the
present study (data not shown). Patterns similar to that determined previously [15] and that found in the present study
have been shown by others [10, 32]. The present study did
record a significant difference in the volume density of the
Sertoli cell in relation to the cycle of the seminiferous epithelium (data not shown). Stages IX through XI showed a
significantly increased percentage of Sertoli cells within the
epithelium, a finding most likely due to the release of the
late spermatids.
Volume of the Seminiferous Epithelium
Also not among the present objectives, but readily obtainable, was the total volume of the seminiferous epithelium, which is shown in Figure 16. The top of this figure
represents the cumulative volumes of all epithelial components and reflects findings similar to the data provided
by Wing and Christensen [16]. Statistical analysis showed
that stages IX-XI were different from most earlier stages
(II-VII). A quick look at Figure 16 reveals that spermiation
is responsible for this decline. The decline, however, is shortlived because there is rapid growth of spermatocytes during the latter part of the cycle.
The data have shown in an objective manner the morphological relationships of the Sertoli cell to the germinal
cells, to the acellular basal lamina and tubular lumen, and
to other Sertoli cells. Volumes of individual germ cell types
are also presented. While the data are largely descriptive,
they allow a better understanding of Sertoli cell structure
and Sertoli-germ cell structural relationships; they also serve
as a basis from which to interpret physiological events and
biochemical data.
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2. Sar M, Hall SH, Wilson EM, French FS. Androgen Regulation of Sertoli cells. In:
Russell LD, Griswold MD (eds.), The Sertoli Cell. Clearwater, FL: Cache River
Press; 1993: 509-516.
3. Sharpe R. Experimental evidence for Sertoli-germ cell and Sertoli-Leydig cell
interactions. In: Russell LD, Griswold MD (eds.), The Sertoli Cell. Clearwater, FL:
Cache River Press; 1993: 391-418.
4. Parvinen M. Cyclic functions of Sertoli cells. In: Russell LD, Griswold MD (eds.),
The Sertoli Cell. Clearwater, FL: Cache River Press, 1993: 331-347.
5. Russell LD. Three-dimensional reconstruction of a rat stage v Sertoli cell: III. A
study of specific cellular relationships. Am J Anat 1983; 167:181-192.
6. Morales C, Clermont Y. Structural changes of the Sertoli cell during the cycle of
the seminiferous epithelium. In: Russell LD, Griswold MD (eds.), The Sertoli
Cell. Clearwater, FL: Cache River Press; 1993: 305-329.
7. Wong V, Russell LD. Three-dimensional reconstruction of a rat stage V Sertoli
cell: I. Methods, basic configuration, and dimensions. Am J Anat 1983; 167:143161.
8. Russell LD, Gardner RJ,Weber JE. Reconstruction of a type B configuration monkey Sertoli cell: size, shape, and configurational and specialized cell-to-cell relationships. Am J Anat 1986; 175:73-90.
9. Sinha Hikim AP, Amador AG, Klemcke HG, Bartke A,Russell LD. Correlative morphology and endocrinology of Sertoli cells in hamster testes in active and inactive states of spermatogenesis. Endocrinology 1989; 125:1829-1843.
10. Bugge HP, Pl6en L. Changes in the volume of Sertoli cells during the cycle of
the seminiferous epithelium in the rat. J Reprod Fertil 1986; 76:39-42.
11. BozzolaiJ, Russell LD. Quantitative electron microscopy. In: Electron Microscopy:
Principles and Techniques for Biologists. Boston: Jones and Bartlett; 1992: 287304.
12. Dym M, Fawcett DW. The blood-testis barrier in the rat and the physiological
compartmentation of the seminiferous epithelium. Biol Reprod 1970; 3:308-326.
13. Russell L. Movement of spermatocytes from the basal to the adluminal compartment of the rat testis. Am J Anat 1977; 148:313-328.
14. Russell LD. The blood-testis barrier and its formation relative to spermatocyte
maturation in the adult rat: a lanthanum tracer study. Anat Rec 1978; 190:99-111.
15. Ye S-J, Ying L, Ghosh S, Franca LR, Russell LD. The Sertoli cell cycle: a re-examination of the structural changes during the cycle of the seminiferous epithelium. Anat Rec 1993; (in press).
16. Wing TY, Christensen AK. Morphometric studies on rat seminiferous tubules. Am
J Anat 1982; 165:13-25.
17. Weber JE, Russell LD, Wong V, Peterson RN. Three-dimensional reconstruction
of a rat stage V Sertoli cell: II. Morphometry of Sertoli-Sertoli and Sertoli-germcell relationships. Am J Anat 1983; 167:163-179.
18. Russell LD. Spermatid-Sertoli tubulobulbar complexes as devices for elimination
of cytoplasm from the head region of late spermatids of the rat. Anat Rec 1979;
194:233-246.
19. Morales C, Clermont Y, Hermo L. Nature and function of endocytosis in Sertoli
cells of the rat. Am J Anat 1985; 173:203-217.
20. Morales C, Clermont Y. Receptor-mediated endocytosis of transferrin by Sertoli
cells of the rat. Biol Reprod 1986; 35:393-405.
21. Russell L. Observations on rat Sertoli ectoplasmic ('junctional') specializations in
their association with germ cells of the rat testis. Tissue &Cell 1977; 9:475-498.
22. Morales C, Clermont Y.Evolution of Sertoli cell processes invading the cytoplasm
of rat spermatids. Anat Rec 1982; 203:233-244.
23. Russell LD. Morphological and functional evidence for Sertoli-germ cell relationships. In: Russell LD, Griswold MD (eds.), The Sertoli Cell. Clearwater, FL:
Cache River Press; 1993: 365-390.
24. Russell L, Clermont Y. Anchoring device between Sertoli cells and late spermatids in rat seminiferous tubules. Anat Rec 1976; 185:259-278.
25. Russell LD. Role in Spermiation. In: Russell LD, Griswold MD (eds.), The Sertoli
Cell. Clearwater, FL:Cache River Press; 1993: 269-303.
26. Russell L, Myers P, OstenburgJ, Malone J. Sertoli ectoplasmic specializations during spermatogenesis. In: Steinberger A, Steinberger E (eds.), Testicular Development, Structure and Function. New York: Raven Press; 1980: 55-69.
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28. Russell LD, Frank B. Characterization of rat spermatocytes after plastic embedding. Arch Androl 1978; 1:15-18.
29. Roosen-Runge EC. Quantitative studies on spermatogenesis in the albino rat. III.
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1955; 25:25-33.
30. Sprando RL, Russell LD. Comparative study of cytoplasmic elimination in spermatids of selected mammalian species. Am J Anat 1987; 178:72-80.
31. Russell LD. Deformaties in the head region of late spermatids of hypophysectomized-hormone-treated rats. Anat Rec 1980; 197:21-31.
32. Kerr JB, Mayberry RA, Irby DC. Morphometric studies on lipid inclusions in Sertoli cells during the spermatogenic cycle in the rat. Cell Tissue Res 1984; 236:699709.