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Postnatal differentiation and development of the rat epididymisA stereological study.

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THE ANATOMICAL RECORD 238~191-198 (1994)
Postnatal Differentiation and Development of the Rat Epididymis:
A Stereological Study
F.X. JIANG, P. TEMPLE-SMITH, AND N.G. WREFORD
Department of Anatomy, Monush University, Clayton, Vic 31 68, Australia
ABSTRACT
Postnatal development and differentiation of the rat epididymis was studied in the rat from 15 to 120 days of life using stereological
techniques. Both the relative volume (volume density) and absolute volume
of the epithelial, interstitial, and luminal compartments in the initial segment, caput, corpus, and cauda epididymides were determined. In all segments the volume density of the epithelial compartment increased between
days 15 and 30 before falling to adult values at 45 days in the initial segment
(0.476 & 0.0311, at 60 days in the caput (0.258 2 0.028) and at 90 days in the
corpus (0.245 -t 0.007) and cauda (0.140 & 0.004). The relative volume of the
interstitiurn decreased, whilst that of the lumen increased over the same
period with adult values being achieved earlier in the proximal segments
than in the distal segments. In contrast to volume fraction the absolute
volume of all compartments in all segments increased from day 15 to day 90.
Between 90 and 120 days the absolute volumes of compartments in the
initial segment and caput showed little volume change. All compartments
in the corpus and cauda showed significant increases in volume over the
same period. A similar pattern of development was observed with respect
to the surface area of both the luminal and basement membrane aspects of
the epithelium; surface area per unit volume (surface density) in all segments reached adult values at approximately 60 days, whilst the increase in
absolute area of the surfaces ceased at 90 days in the initial segment and
caput and continued to 120 days in the corpus and cauda. The total length
of the epididymal tubule showed the same pattern with no increase in
length apparent in the initial segment and caput after 90 days. Length however continued to increase in the corpus and cauda between 90 and 120
days. Tubule and luminal diameter reached their definitive values in the
initial segment on day 60 but continued to increase until day 90 in the distal
segments. Epithelial height increased between 15 and 30 days in all segments; in the caput, epithelial height was greatest on day 30 before decreasing slightly; and in the corpus and cauda maximal epithelial height was
observed on day 45 decreasing to stable values on day 60 and 90,respectively. All the data is consistent with a proximal to distal differentiation and
development of the epididymis with growth continuing in all segments after
the establishment of the definitive architecture. Growth of the distal s e g
ments continued after its cessation in the proximal segments.
0 1994 Wiley-Liss, Inc.
There have been few stereological studies of the rat the spatial distribution of the surfaces. The present
epididymis; Bartsch et al. (1978) were the first to uti- study was undertaken using isotropic uniform random
lise stereology as a tool to describe epididymal struc- sections to investigate the differential and absolute
ture. Subsequently several reports (Aire, 1979; Djak- growth of the various epididymal compartments during
iew and Jones, 1982; Miller and Killian, 1987; Oke et normal development.
al., 1988) have described stereological aspects of the
MATERIALS AND METHODS
epididymis in a variety of species. Common to all these
Animals
studies is the assumption that the classical stereologiPregnant
Sprague-Dawley
rats (17-20 days gestacal methods for surfaces are applicable to the epididyma1 epithelium. Jiang et al. (1992) have recently con- tion) were obtained from the Central Animal House a t
firmed that this methodology is valid in the adult.
However it remains more rigorous to use isotropic uniReceived July 23, 1993; accepted August 17, 1993.
form random sections as it avoids assumptions about
0 1994 WILEY-LISS, INC.
192
F.X. JIANG
Monash University and maintained at a temperature
of 19 t 1"C, 14 h light and 10 h dark with food and
water ad libitum. The day of birth was defined as day
0. Litters were sexed and female pups discarded. Male
pups were pooled into litters of ten for each nursing
mother until use or weaning a t the end of the fourth
week after birth. Groups of five animals were used at
each time point.
Fixation and Tissue Sampling
Epididymides were fixed by perfusion and subsequent immersion with a glutaraldehyde-formaldehyde
fixative (Jiang et al., 1992). Following fixation the
epididymides were divided into an initial segment,
caput, corpus and cauda (Robaire and Hermo, 1988)
and weighed. In older animals (230 days) the segments
were sliced into a series of slabs approximately 1.5 mm
thick, the slabs were then subdivided into bars measuring approximately 1.5 x 1.5 mm2 in cross section;
the bars were arranged end to end and further divided
into blocks measuring 1.5 x 1.5 x 1.5 mm3. Four of
these blocks were then selected for stereological analysis using a systematic uniform random scheme (Gundersen et al., 1988). In the younger animals the segments were divided into three to four blocks and all
blocks were processed. The sampled blocks were postfixed in 2% osmium tetroxide in 0.2 M cacodylate
buffer stained en bloc with uranyl acetate in 0.2 M
maleate buffer, dehydrated in a series of graded concentrations of ethanol, and embedded in Epon-Araldite
resin with no specific orientation. Sections (1 pm) were
cut with glass knives, stained with 1%toluidine blue in
1% borax, and examined using light microscopy. The
specific gravity was determined by estimating the
buoyant density in a series of sucrose solutions (Bertram et al., 1986). The shrinkage due to processing was
determined for representative blocks of each segment
by measuring the tissue volume before and after processing as previously described by Zhengwei et al.
(1990).
Where 'CPinter,
'CPepith,
and 'CPlumare the total number of points hitting interstitial, epithelial, and duct
luminal compartments; CPTot,epid
= CPinter + ZPepith
+ CP,,,; Ilum and
denote intersections between
a test line and the luminal and basement membrane
aspects of the epididymal epithelium; Up) is the length
of the test line associated with a test point hitting the
epididymis, and M is the magnification. The luminal
aspect of the epithelium was defined as the apical margin of the cytoplasm deep to any surface specialisation
(cilia or microvilli). The basement membrane aspect of
the epithelium was defined as the base of the cytoplasm
which is adjacent to the basement membrane (refer to
Fig. 1).
The diameters of both the tubule and its lumen were
calculated from the ratio of the volume to surface density assuming a cylindrical model. The diameter of the
duct and its lumen were also directly measured on a
digitizing tablet using Sigmascan 3.9 (Jandel Scientific, Corte Madera, CA) in conjunction with a Leitz
Orthoplan microscope equipped with a drawing tube.
Since sections of the epididymis contain both circular
and elliptical profiles of the tubule the diameter and
epithelial height were measured across the lesser of the
long and short axes of the profiles. Length density of
the ductus epididymidis was calculated from volume
and the directly measured diameter data assuming a
cylindrical model.
Absolute data (volume, surface, and length) were calculated as the product of the density data and reference
volume of the relevant segment.
Statistics
Data were analysed using the Peritz F test for mulStereologicalAnalysis
Volume density (fraction) of interstitium (VVinterst tiple comparisons (Harper, 19841, programmed on a
computer. Differences were considered to be
epid), epithelium (VVepith,epid),and lumen ( v ~ , ,id)~of, ~ personal
the epididymis together with surface density of tge lu- significant when P < 0.05. Data are reported as mean
minal
(SVlum,epjd)
and basement
membrane t SEM in both tables and figures.
(Sn.mem,epid) aspects of the epitheiium in the rat epiRESULTS
didymis were determined on sections obtained from the
Histological Material
arbitrarily orientated tissue blocks using classical
Fixation of the tissues used in this study was good,
methods for surfaces (Jiang et al., 1992). The section
was located a t low magnification (93 x ) a random field some minor separation artefact was apparent in the
was then examined a t higher magnification (284 x ) interstitium especially in the peritubular regions. Repusing a projection microscope (Leitz Neopromar) and resentative sections are shown in Figure 1.
probed with a coherent square test system [l(p) = 10
Shrinkage and Specific Gravity
mm, a(p) = 100 mm21. Stereological parameters were
Shrinkage of the epididymal segments from collecthen calculated using the following standard formulae
tion (after perfusion) to final sectioning varied from 12
(Weibel, 1979):
to 22% (by volume). There were no significant differences between segments. Specific gravity in all segCPinterst
ments was approximately 1.068 at day 15. It declined
VVinter.epid =
xpTot,epidl
(1 to 2%)to a minimum at day 45 before rising again a t
day 60; it then slowly declined to the value observed at
day 120. In the 120 day animals specific gravity was
1.0593 t: 0.0009 in the initial segment, 1.0578 t
~
DEVELOPMENTAL STEREOLOGY OF RAT EPIDIDYMIS
193
Fig. 1. Representative micrographs of the initial segment of the epididymis from a rat aged 15 days (A)
and the cauda epididymidis from a rat aged 45 (B)days. Epithelial, interstitial, and luminal compartments together with the luminal and basement membrane aspects of the epithelium are readily discerned. Printed magnification is x 200.
0.0010 in the caput, 1.0595 rt 0.0004 in the corpus, and
1.0670 0.0002 in the cauda (P< 0.001 compared to
other segments).
0.031 on day 45 and then remained relatively constant
to 120 days. In the caput it fell rapidly from 0.558 +0.022 on day 30 to 0.258 rt 0.028 on day 60 and remained constant thereafter. In the corpus the fall in
Postnatal Growth of Epididymal Segments
volume fraction was greatest between day 45 (0.476 It
All segments grew rapidly in the period from 15 to 90 0.018) and day 60 (0.328 rt 0.018); it then slowly fell to
days (Table 1) with the most rapid growth occurring 0.220 If: 0.011 on day 120; a similar pattern was obbetween days 15 and 45 (8- to 15-fold);growth was less served in the cauda.
In contrast to the pattern observed with volume fracmarked thereafter. Growth in the proximal segments
had essentially ceased by 90 days but continued in the tion data, growth in absolute volume of the epithelium
distal segments which increased by 1.7- and 1.6-fold (Table 2) was continuous from 15 to 90 days in all segbetween 90 and 120 days in the corpus and cauda, re- ments. Between 90 and 120 days there was no significant change in epithelial volume in the initial segment
spectively.
and the caput but substantial growth was still apparEpithelium
ent in both the corpus (1.5-fold) and the cauda epiThe epithelium was a relatively minor component of didymides (1.75-fold).
all segments of the epididymis at day 15, representing
25 to 31% of the tissue volume (Fig. 2). Rapid differen- lnterstitium
tial growth occurred in this component between 15 and
The interstitium was the dominant compartment in
30 days and a t 30 days it constituted in excess of 5010 all segments of the epididymis at day 15 representing
of tissue volume in the initial segment, caput, and cor- approximately 70% of the tissue volume (Fig. 3). In all
pus; however this growth was less marked in the cauda segments interstitial volume fraction fell as developwhere the epithelium constituted 33%of the tissue vol- ment progressed, this was most marked in the initial
ume on day 30. After day 30 the volume of the epithe- segment where the less vigorous growth, relative to
lium relative to the segmental volume decreased in all that of the whole segment, produced a rapid decrease in
segments (i.e., it grew less than the segment as a the volume fraction from 0.746 rt 0.011 on day 15 to
whole). In the initial segment it decreased from a vol- 0.410 It 0.020 on day 30 and plateaued a t 0.320 rt 0.013
ume fraction of 0.564 +- 0.023 on day 30 to 0.476 It on day 60. A similar pattern was observed in more
194
F.X. JIANG
TABLE 1. Segmental volumes (mm3)of the rat epididymis from day 15 to day 120
Caput
Initial segment
1.37 t 0.17
4.18 t 0.27'
12.4 0.6l
25.5 t 2.6l
37.2 t 3.42
39.9 i 2.9
Day 15
Day 30
Day 45
Day 60
Day 90
Dav 120
2.72 t 0.18
8.37 t 0.74l
41.9 2 3.01
97.1 t 3.9'
173 i 8l
195 10
*
*
corpus
1.42 0.07
6.60 2 0.63l
16.2 t 1.5l
33.1 1.7l
49.5 2 3.71
83.1 t 4.7l
*
*
Cauda
3.90 i 0.47
11.6 i 0.9'
32.5 It 1.3l
67.5 i 7.5l
148 3l
237 t 13l
*
'P < 0.01 vs. the same segment in previous age stage.
'P < 0.05 vs. the same segment in previous age stage.
***
06
E
2
***
06
05
05
04
04
0.3
03
-
50-60% of the epididymis in the caput corpus and
cauda but only 20% in the initial segment.
Luminal volume (Table 4) increased in all segments
up to 90 days with the maximum rate of growth occurring between days 45 and 60 in the initial segment,
caput, and corpus. There was no change in the absolute
volume of the lumen in the initial segment and caput
after 90 days. Maximum rate of growth in the luminal
compartment of the cauda was observed between days
60 and 90 and luminal volume continued to increase in
both the corpus and cauda between 90 and 120 days.
0.7
07
d
0.2
02
(Initial Segment)
-
01
.+.
00
(Caput)
01
W
ll
00
0 15 30 45 60
n
90
15 30 45 60
120
1
~
.... .. .-.
. ..
90
120
O7
06
05
04
03
03
(Corpus)
02
i
0
15 30 45 60
90
120
_ _0
15 30 45 60
90
120
Days after B i r t h
Fig. 2. Epithelial volume fraction as a function of age in each
segment of the rat epididymis from day 15 to day 120. *P < 0.05 and
***P < 0.005 vs previous age stage. Bar is mean t SEM.
distal segments although the rate of change was less
marked with distal progression.
Interstitial volume (Table 3) increased in all segments throughout the study period. However there was
no significant change in volume between 90 and 120
days in the initial segment, whereas there was significant increase in the caput (1.3-fold) and a doubling in
the corpus and cauda.
Lumen
The lumen of the epididymal duct system was minimal and represented less than 2% of the epididymal
volume a t 15 days (Fig. 4). There was a rapid increase
in volume fraction from 30 days; in the initial segment
and caput the majority of the increase occurred between days 30 and 60, with minimal change thereafter.
The volume fraction of the lumen continued to increase
in the more distal regions until 90 days with little
change thereafter. Definitively the lumen constituted
Surface Area of the Luminal Border
The surface density of the luminal border in 15-dayold animals ranged from 2.83 +- 0.91 mm-' (in the
initial segment) to 3.91 rt 0.78 mm-' (in the corpus)
with no significant differences between segments. Surface density increased between 15 and 60 days and was
stable between 8 and 10 mm-' from 60 to 120 days in
all segments.
The total surface area of the luminal border (Table 5 )
in the initial segment increased from 4.18 1.50 mm2
on day 15 to 355 rt 42 mm2 on day 90, with no further
increase to day 120; a similar pattern was observed in
the caput. In contrast in the corpus and cauda the surface area continued to increase to 120 days, in the corpus there was a 48% increase from 491 rt 67 to 727 rt
54 mm2 between 90 and 120 days, while in the cauda
there was a 75% increase 1,024 f 54 to 1,797 rt 193
mm2 in the same period.
Surface Area of the Basement Membrane Aspect
of the Epithelium
Surface density of the basement membrane aspect of
the epithelium, on day 15 was 27.2 rt 1.9 mm-', 26.7 rt
1.1 mm-', 21.0 +- 0.9 mm-l, and 17.1 rt 1.2 mm-' in
the initial segment, caput, corpus, and cauda, respectively. There was little change in surface density from
15 to 30 days, but the period from 30 to 60 days was
characterised by a relatively rapid decline in all segments. Definitively (120 days) surface density was
16.6 rt 1.1 mm-' in the initial se ent, 10.1 f 0.9
mm-' in the caput, 10.6 rt 0.5 m m - c the corpus and
8.40 +- 0.90 mm-' in the cauda.
The total surface area of the basement membrane
(Table 6) showed a continuous increase from 15 to 90
days in the initial segment and caput, there was a further small but not significant increase (22%) in the
initial segment between 90 and 120 days, no change
was observed in the caput over the same period. In the
corpus and cauda there was a continuous increase from
195
DEVELOPMENTAL STEREOLOGY OF RAT EPIDIDYMIS
TABLE 2. Epithelial volume (mm3)of each segment of the rat epididymis from day
15 to day 120
Initial segment
0.336 t 0.038
2.32 t 0.07l
5.97 t 0.64l
12.7 t 1.3l
17.5 t 1.3l
19.3 t 2.8
Dav 15
D& 30
Day 45
Day 60
Day 90
Day 120
Caput
0.808 t 0.01
4.73 t 0.56l
15.8 rt 0.54l
25.3 t 3.2l
44.5 t 4.8l
46.4 t 1.9
corpus
0.438 t 0.054
3.29 t 0.31'
7.60 t 0.501
10.8 rt 0.4l
12.1 t 1.2
18.4 2 1.7l
Cauda
0.956 t 0.110
3.66 rt 0.25l
10.2 t 2.7l
15.8 t 2.2l
20.7 t 0.9
36.1 t 3.9l
lP < 0.01 vs. the same segment in previous age stage
day 90 and 120. In the corpus and cauda total length
increased throughout the period 15 to 120 days with a
significant increase between 90 and 120 days in the
cauda.
0 75
4
0 60
Epithelial Height
\**%
0 45
0 30
0 15
-
p
/,
1
(Initial Segment)
i_ii_iii_i-L
0 0c
"l
0
*
0 15
""
15
10 45 60
90
15 30 45 60
120
90
120
0 60
0 30
(Cauda)
0 15
1
0151
'
0
15 30 45 60
,--AJ
90
000
'20
Epithelial height is readily calculated from the difference between the duct and lumen diameters (Table
7). In the initial segment it increased rapidly from 13.3
rt 2.1 pm to 41.0
3.7 pm between 15 and 45 days and
showed little change thereafter. In the caput it increased from 18.3 It 5.5 pm to 33.7 k 2.0 pm on day 30
followed by a slight but not significant decrease. In the
corpus there was again a rapid increase from 19.1 k 1.7
pm on day 15 to 49.6 k 5.1 pm on 45 followed by a
decrease to 42.7 rt 7.0 pm a t day 60, with stable values
thereafter. A similar pattern was observed in the
cauda with a maximum of 27.4 k 4.7 pm on day 45
decreasing to 18.0 I
7.6 pm on day 90. Direct measurement of epithelial height gave similar values to those
calculated from the density data, the only exception to
this was in the cauda on day 45 when the lumen appears very irregular (Fig. 1B) and direct measurement
is difficult.
0
15 30 45 60
90
I20
Days a f t e r Birth
DISCUSSION
Shrinkage and Specific Gravity
In this work stereological data was estimated in
Epon embedded tissue, whilst reference volume was
determined on the freshly perfused tissue. Absolute
data is reported as the product of the stereological ratio
and the reference volume of the freshly perfused seg15 to 120 days with increases of 46% and 71% respec- ment. Volume shrinkage due to processing was uniform between segments and varied between 12% and
tively between 90 and 120 days.
21%. Bertram et al. (1986) have demonstrated that tisDiameter and Length of the Epididymal Tubule
sue shrinkage varies with tissue type, shrinkage, and
Tubule diameter (Table 7) in the initial segment in- swelling occurring within the same block when multicreased from 37.8 k 2.2 pm at 15 days to 175 k 7 at 60 ple tissue types were present. Bolender (1983) has sugdays with little change between 60 and 120 days. In the gested that volume and surface may well behave difcaput corpus and cauda the diameter increased from 15 ferently during tissue shrinkage since surface can
to 90 days and was stable between 90 and 120 days. accommodate volume shrinkage by becoming crenated.
Definitively the diameter was lowest in the initial seg- Thus the application of shrinkage corrections in a
ment (165 k 11 pm), intermediate in the caput (298 I study concerned with volume and surface area is not
35 pm), and corpus (276 12 pm) and largest in the justifiable. In this work density data derived from procauda (333 k 32 pm). A similar pattern of age depen- cessed tissue have been combined with the reference
dent increase was observed with the luminal diameter. volume measured on freshly perfused tissue to yield
Definitively the luminal diameter was smallest in the absolute volume and surface data. The reporting of the
initial segment (87.1 k 4.0 pm), intermediate in the data for volume change associated with processing will
caput (239 I29 pm) and corpus (232 8 pm), and facilitate the comparison between this and other studies.
largest in the cauda (286 It 38 pm).
Specific gravity peaked on day 30 and fell to a minTotal length of the epididymal tubule (Table 8 ) increased continuously from day 15 to day 90 in the ini- imum on day 45, probably reflecting the formation of
tial segment and corpus, with little change between the tubular lumen. Definitively the specific gravity
Fig. 3. Interstitial volume fraction as a function of age in each segment of the rat epididymis from day 15 to day 120. *P < 0.05 and ***P
< 0.001 vs previous age stage. Bar is mean 2 SEM.
*
196
F.X. JIANG
TABLE 3. Interstitial volume (mm3)of each segment of the rat epididymis from day
15 to day 120
Initial segment
1.02 t 0.13
1.69 i 0.162
5.41 lr: 0.35l
8.10 t 0.94'
11.1i 1.5
12.9 i 0.5
Day 15
Day 30
Day 45
Day 60
Day 90
Day 120
Caput
1.87 i 0.09
3.43 t 0.24'
12.5 t 1.6l
24.6 i 2.8l
38.4 t 5.3'
51.2 t 5.2'
corpus
0.968 i 0.098
3.12 i 0.32l
6.89 rt 0.73l
9.75 t 1.10
11.5 2 1.5
22.8 t 0.9l
Cauda
2.80 t 0.35
7.06 t 0.92l
17.0 2 1.4l
26.2 t 3.7
39.2 t 1.9'
74.7 2 8.5'
'P < 0.01 vs. the same segment in previous age stage.
' P < 0.05 vs. the same segment in previous age stage.
O
0 65
g
3
%
?
i
(Initial Segment)
_I/ ;p
01
00
I&'&,,
0
15 30 45 60
, , ,
90
120
I
0
15
30 45 60
90
120
1
0
**J
15 30 45 60
90
l?O
Days a f t e r Birth
Fig. 4. Luminal volume fraction as a function of age in each segment
of the rat epididymis from day 15 to day 120. **P< 0.01 and ***P <
0.005 vs previous age stage. Bar is mean 2 SEM.
was significantly higher in the cauda than the other
segments on day 120 and this probably reflects the
high sperm content in this region a t this time.
Postnatal Changes in Stereological Parameters
of the Epididymis
In this work both ratio (volume density, surface density, and length density) and absolute data have been
determined. Examination of the volume density data
gives an indication of the development and differentiation of the epididymis, whilst absolute data gives an
indication of its growth. The volume density data reported in this study clearly shows a progressive development from proximal to distal regions of the rat epididymis. Volume density of the epithelium reached
adult levels in the initial segment on day 45, in the
caput on day 60, and in the corpus and cauda on day 90.
Volume density of the lumen also approached adult
levels in the initial segment and caput on day 60 and in
the corpus and cauda on day 90. Growth in the absolute
volume of the initial segment and caput essentially
ceased on day 90, whilst considerable growth occurred
in the corpus and cauda in the period 90 to 120 days. In
all segments the rapid increase in both the absolute
and volume fraction of the luminal compartment in the
peripubertal period (30-60 days) was the dominant
factor influencing epididymal maturation.
In this study surface density estimates were performed on isotropic uniform random sections (IUR)
rather than vertical sections (Baddeley et al., 1986).
The use of vertical sections is the preferred method for
the estimation of surface density. However it is difficult to obtain sections with a well-defined vertical axis
from small irregularly shaped pieces of tissue, such as
the developing epididymis. In a previous study (Jiang
et al., 1992)we have demonstrated using both IUR and
fixed orientations sections that the surface of the epididymal tubule is isotropic. In other studies (unpublished data) on adult animals we have used vertical
sections for surface density estimation and obtained
results consistent with those from IUR sections. In the
present study we used IUR sections to ensure that surface estimates were valid a t all ages. Surface and
length density have dimensions mm-' and mm-2, respectively; changes in either parameter are not as
simple to interpret as changes in volume density which
is dimensionless. In contrast to the density data estimates of total surface area and length are readily interpreted in terms of growth. Surface area of both the
luminal and basement membrane aspects of the epithelium did not change after 90 days in the initial segment and caput but substantial growth occurred
between 90 and 120 days in the corpus and cauda. Surface areas reported in this study are an underestimate;
the true surface area is amplified by the presence of
surface specialisations which have been estimated to
increase the luminal surface area 2.3-fold in the adult
rat (Djakiew and Jones, 1982).
If the epididymal tubule is assumed to be a convoluted cylinder, surface, and length density are simply
functions of volume density and diameter. Diameter
data is absolute and readily interpreted, for a constant
volume density a smaller diameter tubule has a
greater surface density. The diameter of the epididyma1 tubule can be determined by either direct measurement or calculated from the ratio of the volume to
surface density (subject to the assumption of a cylindrical model); in the present study the latter method
was used. There was a close agreement between calculated and directly measured values in all segments of
the epididymis, except in the cauda on day 45 when
epithelial hypertrophy produced irregularities of the
197
DEVELOPMENTAL STEREOLOGY OF RAT EPIDIDYMIS
TABLE 4. Luminal volume (mm3)of each segment of the rat epididymis from day 15
to day 120
Day 15
Day 30
Day 45
Day 60
Day 90
Dav 120
Initial segment
0.008 t 0.004
0.112 t 0.0212
1.04 t 0.17l
4.61 t 0.59l
8.59 t 0.68l
7.68 t 0.87
Caput
0.028 t 0.011
0.226 t 0.046l
13.6 t 1.6l
47.1 t 4.7l
92.9 t 7.5l
97.6 t 14.0
corpus
0.016 t 0.007
0.176 t 0.047l
1.68 t 0.401
12.6 t l.ll
25.9 t 2.01
42.0 t 2.6l
Cauda
0.082 t 0.035
0.870 t 0.16l
5.32 t 0.38l
25.6 t 2.3l
87.8 t 2.5l
126 t 15l
' P < 0.01 vs. the same segment in previous age stage.
' P < 0.05 vs. the same segment in previous age stage.
TABLE 5. Surface area (mm') of the luminal border of each segment of the rat
epididymis from day 15 to day 120
Day 15
Day 30
Day 45
Day 60
Day 90
Dav 120
Initial segment
4.18 t 1.50
22.7 t 3.9l
79.6 t 8.5l
215 t 15l
355 t 42l
350 t 35
Caput
10.4 rt 1.7
45.8 t 5.6l
433 2 32l
903 t 401
1,627 t 134l
1.620 t 52
corpus
5.79 t 1.60
30.6 t 5.2l
105 t 18l
320 t 16l
491 t 672
727 rt 54'
Cauda
11.9 t 2.1
45.7 t 4.41
144 t 26l
542 2 56l
1,024 t 54l
1.797 t 193l
'P < 0.01 vs. the same segment in previous age stage.
2P< 0.05 vs. the same segment in previous age stage.
TABLE 6. Surface area (mm') of the basement membrane aspect of the epithelium of
each segment of the rat epididymis from day 15 to day 120
Day 15
Day 30
Day 45
Day 60
Day 90
Day 120
Initial secrment
36.7 t 3.9
111 t 8l
212 t 27l
402 t 52l
542 rt 51
660 rt 57
Camt
72.3 t 3.1
228 t 26l
675 t 8l
1,153 t 10l1
1,917 t 2552
1,957 t 81
Comus
29.7 t 3.8
136 t lo1
227 t 19l
386 t 22l
606 t 68l
882 rt 8l
Cauda
67.2 rt 11
172 t 8l
349 t 16l
682 t 1101
1,152 t 87l
1,974 t 177l
lP < 0.01 vs. the same segment in previous age stage.
'P < 0.05 vs. the same segment in previous age stage.
TABLE 7. Tubular and luminal diameters (pm) of the ductus epididymis calculated from surface density and
volume fraction data
Day 15
Day 30
Day 45
Day 60
Day90
Day 120
Initial segment
Tubule
Lumen
8.6 t 4.4
37.8 t 2.2
87.8 t 1.8l 22.3 t 4.8l
51.4 t 5.3l
133 rt 6l
85.2 t 9.2'
175 t 7l
194 t 7
98.6 t 7.6
165 t 11
87.1 t 4.0
Caput
Tubule
46.6 t 3.6
86.8 t 2.2l
174 t 111
258 t 23'
294 t 20
298 5 35
Lumen
9.9 t 1.8
19.4 t 2.4l
124 t 8l
212 t 25l
229 rt 17
239 t 29
corpus
Tubule
Lumen
16.8 2 6.9
62.2 t 3.0
24.8 t 7.6
102 t 4l
64.7 t 10'
164 2 6l
160 t 20'
246 rt 19l
217 rt 18'
256 rt 18
276 2 12
232 2 8
Cauda
Tubule
63.4 It 3.0
105 t 4l
178 rt 5l
255 t 28l
380 2 25'
333 2 32
Lumen
27.0 rt 11.0
74.4 t 9.1'
168 t 301
193 t 18
345 t 111
286 t 38
'P < 0.01 vs. the same segment in previous age stage.
'P < 0.05 vs. the same segment in previous age stage.
tubule lumen which influenced the estimation of it's
diameter. All segments of the epididymis increased in
diameter of both the luminal and basement membrane
aspects of the tubule from 15 to 90 days, with little
change between 90 and 120 days. Between 60 and 90
days the increase in luminal diameter was 12% (not
significant) in the initial segment, 22% in the caput,
37% in the corpus, and 74%in the cauda. This is again
consistent with a progressive differentiation from proximal to distal segments. Definitively the luminal diam-
eter was smallest (high luminal surface to volume ratio) in the proximal segment consistent with a
transport function and highest in the distal segments
consistent with a storage function.
Epithelial height showed a consistent pattern in the
more distal segments; a small peak at 30 days in the
caput decreased to the mature value a t day 45. In the
corpus and cauda it peaked on day 45 before decreasing
to mature values on days 60 and 90, respectively. Definitively epithelial height was greatest in the proxi-
198
F.X.JIANG
TABLE 8. Length of the duct (mm) of each segment of the rat epididymis from day
15 to day 120
Day 15
Day 30
Day 45
Day 60
Day 90
Day 120
Initial segment
374 t 45
476 t 55
579 rt 63
884 rt 80’
1,201 t 128
1,141 rt 81
Caput
702 rt 204
1,041 rt 105
1,259 rt 86
1,943 rt 183l
2,597 rt 342
2,466 t 279
corpus
265 +- 34
505 t 57
533 t 53
727 rt 432
754 rt 90
1,065 t 87
Cauda
504 t 99
491 t 52
662 rt 81
1,117 rt 129l
1,350 2 141
1,938 2 111’
‘P < 0.01 vs. the same segment in previous age stage.
’P < 0.05 vs. the same segment in previous age stage.
ma1 segment, consistent with a transport function and
lowest in the distal segments consistent with a storage
function.
Cell number and volume are important indicators of
organ maturation which have not been addressed in
the present study. The development of the “Optical Disector,” (Braendgaard et al., 1989) which is applicable
to thick methacrylate sections, will facilitate the derivation of such data. It would be appropriate in a future
study to combine stereology with either tritiated thymidine uptake or stathmokinetic techniques to yield
data on cell proliferation and cell number, particularly
in the epithelial compartment. Such data would clarify
the question of when and if growth changed from a
hyperplastic to a hypertrophic mode.
Although there are no other developmental stereological studies of the rat epididymis, it is of interest to
compare these observations with other work. Sun and
Flickinger (1979) investigated the qualitative changes
in the rat epididymis with age. Cytological differentiation was the major feature of the period from 15 to 45
days, whereas the period from 45 to 90 days represented a period of expansion without further differentiation.
To summarise, this report is the first to quantitatively describe the development of the epididymal segments using assumption-free stereological techniques.
The major observation is that the definitive tissue architecture of the epididymis is established in a progressive way from proximal to distal segments with growth
continuing in the distal segments after its cessation in
the proximal segments. This study provides a quantitative basis for the study of this organ.
ACKNOWLEDGMENTS
The authors would like to thank Professor Hans Jorgen Gundersen for reviewing the manuscript and adding clarity to the exposition of the stereological content.
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