Effects of testosterone enanthate on the structure of the male reproductive tract of the rat.

Effects of Testosterone Enanthate on the Structure of the
Male Reproductive Tract of the Rat
CHARLES J. FLICKINGER
Department ofAnatomy, University of Virginia School of Medicine,
Charlottesville, Virginia 22908
ABSTRACT
The histology and fine structure of the testis, epididymis and
sex accessory glands were studied in young adult male rats administered testosterone enanthate, 120 pg/lOO g body weight, three times weekly for 4, 8, or 12
weeks. The weights of the testis and epididymis decreased, and animals treated
for 11 weeks were infertile. Alterations were found in the seminiferous tubules
of all rats treated for 8 or 12 weeks, including the presence of many degenerating germ cells and a large decrease or absence of late spermatids. Study of different stages of the cycle of the seminiferous epithelium showed that the
greatest number of degenerating germ cells, step 7 spermatids and pachytene
primary spermatocytes, occurred a t stages VII-VIII of the cycle. Some normal
appearing spermatogonia, primary spermatocytes and early spermatids remained in most seminiferous tubules. Sertoli cells contained many lipid droplets and lysosome-like bodies, and degenerating cells were surrounded by Sertoli cell cytoplasm. The Leydig cells of treated animals were greatly reduced in
size. Sperm progressively disappeared from the lumen of the middle segment
and proximal part of the terminal segment of the epididymis after treatment
for 8 or 1 2 weeks. Changes in the middle segment also included the appearance
of intraepithelial cavities containing debris, and the presence within the epithelium of phagocytic cells that resembled leukocytes. The lumen of the proximal part of the terminal segment was often collapsed, while in the distal part
of the terminal segment, the lumen was filled with cellular debris and degenerating sperm. Organelles of the principal cells of the epididymal epithelium
appeared to be qualitatively unaltered. The weight of the sex accessory glands
remained close t o normal, and the presence of normal ultrastructural features
suggested that production of secretions continued.
Testosterone is among the hormones and
other compounds considered a s possible
chemical contraceptives because i t is capable
of suppressing spermatogenesis in men
(Hotchkiss, '44; Heller et al., '50, '70; Reddy
and Rao, '72; Steinberger and Smith, '77). The
basis of its antifertility action is a biphasic
action on the seminiferous tubules of several
mammalian species (Albert, '61; Ludwig, '50;
Steinberger, '71; Desjardins et al., '73; Walsh
and Swerdloff, '73; Ewing et al., '73a; Berndtson et al., '74). In relatively low doses i t suppresses spermatogenesis, apparently by decreasing the secretion of gonadotropins by the
pituitary gland (Ludwig, '50; Desjardins et
al., '73; Ewing et al., '73a, '73b; Heller et al.,
'70; Berndtson et al., '741, thus greatly lowerANAT. REC. (1978)192: 555-584.
ing the secretion of endogenous testosterone
by the Leydig cells of the testis. At relatively
high dose levels, however, the exogenous testosterone is itself sufficient for spermatogenesis to continue even though pituitary gonadotropin secretion is depressed (Nelson and
Merckel, '37; Ludwig, '50; Clermont and
Harvey, '65; Walsh and Swerdloff, '73; Desjardins et al., '73; Berndtson et al., '74). The
reason that low doses of testosterone, adequate t o maintain the sex accessory glands
and libido, are insufficient to maintain spermatogenesis probably lies in the anatomical
location of the seminiferous tubules close t o
the source of endogenous testosterone in the
Received Dec. 7, '77. Accepted July 11, '78.
555
556
CHARLES J . FLICKINGER
Leydig cells. Thus t h e seminiferous epithelium normally is exposed t o a much greater
concentration of testosterone than t h e rest of
the body, and this is not replaced by administration of amounts of hormone sufficient for
other androgen-dependent tissues (Heller e t
al., '70; Walsh and Swerdloff, '73). Therefore,
testosterone in proper amount potentially can
result in suppression of spermatogenesis with
maintenance of other male characteristics.
Various testosterone derivatives have been
considered for use in suppressing sperm production, and t h e testosterone ester, testosterone enanthate, has attracted attention because i t has a relatively long action and needs
to be administered less frequently than many
other androgens such as testosterone or testosterone propionate (Murad and Gilman, '75;
Steinberger and Smith, '77). In t h e present
study, the histology and fine structure of t h e
male reproductive tract of rats administered
testosterone enanthate were studied. An objective was t o extend morphological observations on t h e testis t o determine in detail t h e
cell types and stages of t h e cycle of t h e seminiferous epithelium t h a t a r e affected, as well
as to define ultrastructural aspects of t h e alterations. It is also important to consider t h e
sex accessory organs in studies of antifertility
agents. First, under some conditions sperm
production may be decreased but not abolished, and t h e sex accessory organs, especially
t h e epididymis, may affect t h e fate of t h e
sperm t h a t continue t o be produced and t h u s
contribute to t h e antifertility action of t h e
drug. Second, t h e maintenance of t h e sex
accessory glands, secondary sex characteristics, and libido a r e important objectives in t h e
eventual development of practical chemical
contraceptives for men. Therefore, further
objectives were t o determine if there are
changes in several segments of t h e epididymis
and to investigate whether t h e structure of
t h e sex accessory glands is maintained or altered.
MATERIALS A N D METHODS
Young adult male Sprague-Dawley rats
weighing -200 g were obtained from Hilltop
Lab Animals, Scottdale, Pennsylvania. They
were maintained in t h e University Vivarium
in a n air conditioned room and were furnished
food and water ad libitum. Experimental animals were administered testosterone enanthate (Delatestryl, Squibb) 120 pg/lOO g body
weight in 0.1-0.3 ml sesame oil three times
weekly by subcutaneous injection. Control
rats were administered t h e oil vehicle alone.
Three treated and two control animals were
studied after treatment for 4, 8, or 12 weeks.
Three rats were permitted to recover for 8
weeks following treatment for 12 weeks. Tissue was also obtained from several normal
adult male rats for comparison with treated
and control animals. Sprague-Dawley rats
were used so t h a t t h e results could be compared readily with our previous studies of
other antifertility agents (Flickinger and Loving, '76; Loving and Flickinger, '76; Flickinger, '77a,b,c) which were carried out on this
strain.
The rats were killed by cervical dislocation,
and t h e testes, epididymides, ventral prostate
a n d seminal vesicles were removed a n d
weighed. Whole testes were immersed for one
hour in a glutaraldehyde, formaldehyde, and
picric acid fixative (It0 and Karnovsky, '681,
prepared by mixing 8.5 ml of Karnovsky's fixative (Karnovsky, '65) with 1.5 ml of a saturated solution of picric acid and 10 ml of 0.1 M
cacodylate buffer, pH 7.3. After one hour,
pieces were cut from t h e testes, diced, and
fixed for a n additional one hour. Portions of
t h e initial, middle, and terminal segments of
t h e epididymis (Glover and Nicander, '71)
were dissected. The terminal segment was
sub-divided into two parts, one corresponding
to t h e proximal part of t h e cauda which has
t h e smaller diameter (regions 5B and 6A of
Reid and Cleland, '57) and t h e other comprising t h e distal part of t h e cauda (region 6B of
Reid and Cleland). The epididymal segments
were immersed in Karnovsky's fixative (Karnovsky, '651, and pieces of t h e ventral prostate
were placed in t h e glutaraldehyde, formaldehyde, and picric acid fixative. After one hour
t h e samples of epididymis and prostate were
diced and fixed for a n additional one hour. The
seminal vesicles were diced and placed for two
hours in Karnovsky's fixative. After fixation
for a total of two hours in aldehyde, all t h e tissue blocks were rinsed with 0.1 M cacodylate
buffer and post-fixed for one hour in 1%OsO,
in 0.1 M cacodylate buffer at pH 7.3. The tissue
was dehydrated in a graded series of ethanols
followed by propylene oxide, and was embedded in Araldite.
Sections 1 p m thick for light microscopy
were cut with glass knives, mounted on slides,
and stained with 0.5% toluidine blue in 0.5%
sodium borate. Light micrographs were obtained with a n Olympus photomicroscope.
557
EFFECTS OF TESTOSTERONE
Thin sections showing silver t o pale gold interference colors were cut with a diamond knife,
mounted on uncoated grids, and stained with
lead citrate (Reynolds, '63). The preparations
were examined and photographed using a
Philips EM-300 or an AEI EM-801 electron
microscope.
To supplement visual impressions of
changes in the size of Leydig cells of the testis,
the following semiquantitative method was
employed t o estimate the sizes of profiles of
Leydig cells in sections for light microscopy
(Flickinger and Loving, '76; Flickinger, '77a).
Regions of accumulation of interstitial tissue
between three or more seminiferous tubules
were chosen a t random for study. Within such
a region, the length and width of all the cells
identifiable as Leydig cells were measured
using a Zeiss optical micrometer. Although
the shape of the cells varied, a rough estimate
of the area of a cell represented within a section was obtained by assuming the profiles t o
be elliptical and multiplying the length/2 x
width12 x T.Twenty-five cells were measured
for each of the two rats treated for 12 weeks
and for two control animals. The mean areas
of Leydig cells in sections of treated and control rats were compared using a T test.
The fertility of three animals treated for 11
weeks with testosterone enanthate and two
accompanying control rats was tested by caging each male rat for five days with two
female rats. During this time, the females
were checked daily for a vaginal plug, and one
week later they were examined for the presence of fetuses.
RESULTS
Testis
The microscopic structure of the testis of
control rats did not differ from normal. Semi-
niferous tubules contained Sertoli cells and
germ cells in various stages of development
(fig. 1).Degenerating germ cells were infrequently encountered in normal or control rats.
As reported by Russell and Clermont ('77),
those present were usually in stages VII-VIII
or I of the cycle, and generally no more than
one was observed in a cross section of a seminiferous tubule.
The weight of the testis declined to less than
half of control values after treatment with
testosterone enanthate for 8 or 12 weeks
(table 1).Microscopic alterations were slight
or were undetected in the testes of rats
treated for 4 weeks. All rats showed microscopic changes after 8 or 12 weeks, although
the severity of the testicular alterations
varied somewhat from one animal to another.
Testicular alterations visible in the light microscope (figs. 2, 3) included the presence of
many degenerating germ cells, a large decrease in number or absence of late spermatids, especially those of the maturation
phase, and a large decrease in the size of the
Leydig cells of the interstitial tissue.
Degenerating cells
Degenerating cells were identified in light
microscopic sections (figs. 2, 3) by their intense staining with toluidine blue, granular
appearance, and in some instances, separation
from surrounding cells by a clear space. As
many as 10 t o 15 degenerating cells were present in some profiles of the seminiferous
tubules. Some of the degenerating cells were
identified as early spermatids because of their
round shape, location among other early spermatids, and a remnant of the acrosomal cap.
Others appeared to be primary spermatocytes
because of their large size, round shape, and
location in the basal part of the seminiferous
TABLE 1
The weights of reproductive organs and the final body weights of rats treated with testosterone enanthate. The average
weights are shown in g/IOOg body weight and in percent of the corresponding control. The values for
the testis, epididymis and seminal vesicle include the two organs from each of
three treated animals and two control rats at each interval
Time
weeks
Treated
Control
8 Treated
Control
12 Treated
Control
4
Testis
Epididymis
Seminal vesicle
gI100 g
% control
gI100 g
80
0.09
75
0.05
37
0.12
0.08
0.13
62
47
0.07
58
g/100 g
% control
0.36
0.45
0.16
0.43
0.16
0.34
0.12
Y,
g/lOO g
%, control
71
0.11
0.10
0.11
0.11
0.17
0.13
110
0.07
0.07
0.06
0.08
0.10
Ventral prostate
control
117
80
100
131
Body
weight
g
427
423
48 1
509
538
564
558
CHARLES J . FLICKINGER
TABLE 2
epithelium alongside normal-appearing primary spermatocytes. Degenerating germ cells
A comparison o f the number of degenerating cells at
were much more numerous in some profiles of
different stages of the cycle o f t h e seminiferous
epithelium in a rat treated with testosterone
seminiferous tubules than others, so an a t enanthate f o r 12 weeks
tempt was made to relate the presence of degenerating cells to stages of the cycle of the
Degenerating
Degenerating
cells per
Number of
seminiferous epithelium.
Stage
cells per cross
100 Sertoli
Sertoli
Various combinations of cells in sections of
section
nuclei
nuclei
the seminiferous tubules of treated animals
A
1
4.1
345
made it evident that different stages of the cyB
1.2
6.2
291
cle of the seminiferous epithelium were repreC
8.3
38.8
299
sented. Due to the scarcity of late spermatids
D (total)
3.5
16.2
214
D,
7.5
32.6
and their absence from many profiles of
D2
1.1
4.4
tubules we were not able precisely t o identify
all of the stages of the cycle. However, in a
study on the development of the seminiferous
epithelium in immature rats, Clermont and tially underwent degeneration in rats treated
Perey ('57) described a way to divide the cycle with testosterone enanthate.
At the electron microscope level, cells were
of the seminiferous epithelium into four broad
stages based on the appearance of sper- judged to be degenerating if they had an
matogonia and spermatocytes. In this scheme unusually dense cytoplasm, distorted and disthe four broad stages, here designated stages rupted membranous organelles, and an abnorA through D, correspond t o the conventional mal degree of clumping and high density of
14 stages (Leblond and Clermont, '52) as fol- nuclear chromatin. Many of the degenerating
lows: Stage A = stages I - IV; Stage B = cells were identified as spermatids of approxstages IV - VI; Stage C = stages VII - VIII; imately stages 6-8 because of the persistence
of the characteristically shaped acrosome (fig.
Stage D = stages IX - XIV.
Degenerating cells appeared particularly 4).Other degenerating cells, which lay deep in
numerous in Stage C (VII - VIII) with some the seminiferous epithelium close to the basal
also present in Stages B and D. To confirm this lamina, were more difficult to identify on the
impression, sections from one animal treated basis of their ultrastructure, but probably corfor 12 weeks were used for a further semi- responded to the degenerating primary sperquantitative analysis. The numbers of degen- matocytes observed with the light microscope.
erating spermatocytes and early spermatids The scarcity or absence of late spermatids sugwere counted in cross or slightly oblique sec- gested that degeneration of these cells might
tions of seminiferous tubules in the four main have occurred also. Some dense elongated instages of the cycle. The persistence of some clusions in the cytoplasm of Sertoli cells (fig.
acrosome phase spermatids in this animal 5) resembled the condensed nuclei of late spermade it possible in most cases to subdivide matids and thus may have been derived from
Stage D further into two parts designated D, degenerating spermatids.
(stages IX t o XI) and D, (stages XI1 to XIV).
As shown in table 2, by far the largest number Other components of the seminiferous
epithelium
of degenerating cells was present in stage C,
which includes the conventional stages VII
Alterations were not detected in the sperand VIII. The next highest number of degen- matogonia of treated animals. Primary spererating cells was found in Stage D, and sub- matocytes in different stages of meiotic prodivision of this stage showed that most were phase were abundant in all the specimens and
present in the early portion of the stage (D,) many had normal ultrastructural features inwhich immediately follows Stage C. Germ cluding a large nucleus containing synapcells present in Stage C of the cycle in addition tonemal complexes. Although numerous det o spermatogonia include pachytene primary generating early spermatids were identified,
spermatocytes, steps 7-8 early spermatids, and large numbers of Golgi and cap phase sperstages 18-19 late spermatids. Thus light mi- matids (fig. 5) remained and had a normal
croscopic observations suggested that germ morphology. As described above, later spercells a t these stages of development preferen- matids were scarce, and those present usually
~~
~~
~
~~
559
EFFECTS OF TESTOSTERONE
occurred singly rather than in the usual
groups.
Large lipid droplets were present in the cytoplasm of Sertoli cells (fig. 5). These occurred
in most tubules regardless of the stage of the
cycle, in contrast to those that are normally
found a t stage IX (Niemi and Kormano, '65;
Kerr and deKretser, '75). Degenerating cells
usually were found in a space bounded by processes of Sertoli cytoplasm (fig. 41, and the Sertoli cells contained prominent polymorphous
lysosome-like structures which sometimes enclosed remnants of degenerating cells (fig. 5).
The remaining ultrastructural features of the
Sertoli cells did not appear to be altered. In
some specimens in which the spermatid population was severely depleted, processes of Sertoli cells occupied much of the epithelium
near the lumen of the seminiferous tubules.
Leydig cells
Upon inspection of light microscope sections, it was evident that the size of Leydig
cells in the interstitial tissue of the testis was
greatly reduced in all the treated animals. The
mean area of Leydig cell profiles in animals
treated for 1 2 weeks was 22.8 pm2,only 33%of
the control value, which was 70.1 pm'. This
difference was highly significant (p < 0.001).
At the ultrastructural level, cytoplasmic organelles of Leydig cells were greatly reduced
in abundance in treated animals (fig. 7). For
example, the smooth endoplasmic reticulum,
which is known to be involved in testosterone
production (Christensen, '751, occupied much
of the remaining cytoplasm but did not approach the large fields containing this organelle in sections of normal cells (fig. 6).
Fertility
The objectives of this study were t o define
the structural changes of the male reproductive tract in the presence of testosterone
enanthate. However, some information on fertility was also obtained and, although limited,
i t is reported here because i t is of interest
in relation to the morphological alterations.
When the rats subsequently killed a t 1 2 weeks
were tested during the eleventh week of treatment, all of the treated males mated, as indicated by the presence of vaginal plugs in each
of the females with which they were caged,
but none impregnated any females. In contrast, each of the control rats impregnated
both females with which they were caged. His-
tological observations on the reproductive
organs of animals allowed to recover for 8
weeks following 12 weeks treatment showed a
return to normal appearance of the reproductive organs, including the presence of many
late spermatids in the testes.
Epididymis
The structure of the epididymis of control
rats did not differ from observations and previous descriptions of this organ in normal rats
(e.g., Hamilton, '75; Flickinger, '77b,c). Prominent features of the columnar principal cells
included apical microvilli, vesicles, vacuoles
and lysosomes, a large supranuclear Golgi apparatus, and both smooth and rough endoplasmic reticulum. The principal cells were accompanied throughout the duct by small basal
and halo cells. In the initial segment apical
cells were present, while light cells appeared
more distally. The epithelium decreased in
height along the length of the epididymis.
Some additional features of the different segments of the normal epididymis will be noted
when pertinent to the descriptions of treated
animals.
The weight of the epididymis decreased to
58% that of the control following treatment
with testosterone enanthate for 8 or 1 2 weeks
(table 1). Microscopic alterations were small
or undetectable in the epididymides of animals treated with testosterone enanthate for
4 weeks, but those of rats treated for 8 or 12
weeks all showed changes.
Initial segment
The initial segment of the epididymis is
normally lined by a tall pseudostratified columnar epithelium. The small lumen contains few sperm compared with more distal
parts (Glover and Nicander, '71). Consistent
changes from this pattern were not detected
in the initial segment of testosterone enanthate-treated rats a t either the histological or
ultrastructural level.
Middle segment
In the middle segment a pseudostratified
columnar epithelium normally surrounds a
large lumen containing many sperm (fig. 8 ) .
The first change in the middle segment of the
epididymis in treated rats was the reduction
and disappearance of the luminal sperm (fig.
9). In the earlier stages of this change, a t 8
weeks, the sperm were seemingly supplanted
560
CHARLES J. FLICKINGER
in the lumen by a variable number of small
round cells which had features reminiscent of
spermatocytes and early spermatids and did
not contain phagocytosed sperm.
A conspicuous alteration in t h e middle segment after treatment for 12 weeks was t h e appearance within t h e epithelium of large circular or oval cavities many p m in diameter (fig.
9), which were located extracellularly and
were bounded by several principal cells (fig.
10). Microvilli on t h e surfaces of t h e principal
cells projected into t h e space. At t h e periphery, a fine flocculent content was common,
while t h e center contained solid material, including small membrane-bound pieces of cytoplasm, granules, and dense fibers similar t o
those of sperm tails. The intraepithelial cavities appeared to be separate from the epididymal lumen but their morphology afforded
no clues as to how they developed.
Some cells resembling leukocytes are normally present in all segments of the r a t epididymis and have long been referred t o as
"halo" cells (Reid and Cleland, '57; Hoffer et
al., '73). I t has been reported t h a t most of t h e
halo cells in t h e epithelium of t h e r a t male excurrent duct system a r e small lymphocytes
(Dym and Romrell, ' 7 5 ) .The epithelium of t h e
middle segment of t h e epididymis of rats
t r e a t e d with testosterone e n a n t h a t e contained m a n y p a l e - s t a i n i n g cells t h a t r e sembled leukocytes (figs. 11, 12), but differed
from halo cells seen in normal and control rats
in two main ways. First, they occurred not
only singly, but also in aggregates of up t o
about 10 to 12 cells (fig. 131, whereas halo cells
normally occur singly. Second, t h e cells of
treated animals contained many structures
with the polymorphous content of phagocytic
vacuoles (figs. 11, 12). The vacuoles varied in
size from a few tenths of a micrometer up to
large spherical bodies several micrometers in
diameter which occupied most of the cytoplasm of a cell within a plane of section. No obvious relationship between the pale-staining
cells and t h e intraepithelial cavities was
noted, and these cells were not observed in t h e
lumen. In some cases, pale-staining cells, lacking phagocytic vacuoles, were found in t h e
connective tissue and in t h e smooth muscle
coat surrounding t h e epididymal epithelium.
The principal cells of t h e middle segment
had a normal complement of organelles and
did not appear altered, with t h e exception of
their unusual arrangement around t h e intraepithelial cavities described above.
Terminal segment, proximal part (proximal
cauda epididymidis)
The proximal cauda epididymidis is normally lined by a pseudostratified columnar epithelium of medium height, which is composed
mainly of principal cells and light cells. The
smoothly-contoured epithelium surrounds a
lumen containing many sperm (fig. 14). Beginning in rats treated for 8 weeks, sperm progressively disappeared from t h e lumen of the
proximal cauda epididymidis. Concomitant
with t h e loss of sperm, t h e lumen of t h e proximal cauda collapsed and t h e epithelium
assumed a n irregular, undulating contour
(fig. 15).
As in t h e case of t h e middle segment, the
epithelium of the proximal cauda of animals
treated for 12 weeks contained abundant cells
provisionally identified as leukocytes, including aggregates of these pale-staining cells. In
many specimens, particularly those from rats
treated for 8 weeks, t h e light cells of t h e epithelium were very conspicuous (figs. 15) with
their content of apical vesicles and vacuoles
and large numbers of membrane-bound dense
bodies believed to be lysosomes (Nicander, '70;
Flickinger, '72). The principal cells of t h e
proximal cauda epididymidis did not appear to
differ from those of control or normal rats.
Terminal segment, distal part (distal cauda
epididymidis)
The distal part of t h e terminal segment of
t h e normal rat epididymis has a large lumen
containing many sperm, surrounded by t h e
circular profile of a smoothly contoured, low
columnar epithelium (fig. 16). Changes were
observed in t h e luminal content of the distal
cauda epididymidis in one rat treated for 8
weeks and in all those studied after 12 weeks
treatment. In these specimens, t h e entire
lumen was filled with a mass of flocculent material, spheres of cytoplasm, cellular debris,
and some sperm (figs. 17, 18). Most of the
sperm were judged t o be disintegrating by t h e
absence of plasma membranes, frequent distortion of t h e cell organelles, and changes in
t h e relations of organelles t o one another.
There were no regions within t h e lumen t h a t
contained groups of normal sperm as in the
lumen of normal and control rats. Alterations
were not detected in t h e epithelium of t h e distal cauda epididymidis.
Sex accessory glands
The weights of t h e seminal vesicles and ven-
EFFECTS OF TESTOSTERONE
tral prostate gland of animals treated with
testosterone were maintained a t or slightly
above control values (table 1). In accord with
maintenance of t h e weights of t h e organs, t h e
height of t h e glandular epithelium of treated
animals was similar to t h a t of normal and control rats, and histological features of t h e
glands were normal (figs. 19, 20).
At t h e ultrastructural level, both glands
displayed abundant cisternae of rough endoplasmic reticulum, a large Golgi apparatus,
and many secretory vacuoles (figs. 21, 22).
Thus the organelles known to be involved in
t h e synthesis and transport of secretory proteins in both t h e r a t prostate and seminal vesicles (Flickinger, '74a,b) were present and had
a normal morphology, suggesting t h a t there
was a n active secretory process in these
glands.
DISCUSSION
Testis
The results showed t h a t in rats administered testosterone enanthate testis weight declined and spermatogenesis was greatly suppressed. This confirmed earlier reports, based
on light microscopic studies, t h a t testosterone
in various forms and with different routes of
administration can suppress the seminiferous
epithelium of t h e r a t (Abdi and Hasan, '73;
Berndtson e t al., '74; Walsh and Swerdloff, '73).
In t h e present study, observations of t h e effects of testosterone enanthate on t h e testis
were extended to show t h a t certain stages of
germ cells were ereferentially affected; many
degenerating spermatids of stages 6-7 were
definitely identified, and pachytene primary
spermatocytes probably degenerated as well.
Accordingly, degenerating germ cells were
most prevalent in seminiferous tubules corresponding to stages VII-VIII (Leblond and
Clermont, '52) of t h e cycle of t h e seminiferous
epithelium.
The stages of degenerating germ cells observed in testosterone enanthate-treated rats
closely resemble those t h a t degenerated in increased numbers after hypophysectomy in t h e
r a t (Clermont and Morgentaler, '55; Russell
and Clermont, '77). Suppression of spermatogenesis by testosterone treatment has usually
been attributed t o decreased gonadotropin secretion (Ludwig, '50; Desjardins e t al., '73;
Ewing et al., '73a,b; Heller e t al., '70; Berndtson e t al., '74). The present observation t h a t
Leydig cells were greatly reduced in size and
in content of smooth endoplasmic reticulum is
561
in accord with this, and suggests t h a t decreased gonadotropin and endogenous testosterone secretion form t h e basis for t h e similarity between t h e effects of testosterone
enanthate and hypophysectomy on t h e seminiferous tubules. The effects of testosterone
enanthate on t h e testis also closely resemble
t h e alterations observed following treatment
with other antifertility compounds, at least
some of which probably act by suppressing gonadotropin stimulation of t h e testis (Flickinger, '77a,c). In a recent study, Dym and
Madhwa Raj ('77) described changes in t h e
testes of rats administered anti-LH serum.
There was a decrease in serum and rete testis
fluid testosterone and in t h e smooth endoplasmic reticulum of Leydig cells. Changes in the
seminiferous tubules similar to those in t h e
present study comprised degeneration of midpachytene spermatocytes, phagocytosis of
germ cells by Sertoli cells, and a n increase in
Sertoli cell lipid droplets. The rapidity of alterations in Sertoli cells, including the retention of late spermatids and changes in smooth
endoplasmic reticulum, nuclei, and mitochondria, suggested t h a t alterations in germ cells
were secondary to those in Sertoli cells (Dym
and Madhwa Raj, '77).
Although not directly comparable t o t h e
present study because of differences in dosage and in t h e strain of animals used, it is
pertinent t o note t h a t physiological observations on testosterone enanthate-treated rats
(Walsh and Swerdloff, '73) revealed decreased
serum LH levels while serum FSH levels were
not altered. In addition, clinical studies have
shown t h a t testosterone enanthate results in
suppression of Leydig cells and spermatogenesis in men (Heller e t al., '70; Steinberger and
Smith, '77), and i t has been proposed t h a t
these effects are mediated through suppression of LH (Heller et al., '70). The situation
may be more complicated in primates than in
rats and rabbits, however, since testosterone
administration via subdermal implantation in
Rhesus monkeys reduced sperm production
while serum LH levels remained normal
(Ewing et al., '76).
The seminiferous tubules were altered in the
presence of exogenous testosterone enanthate
sufficient to maintain t h e weight and fine
structure of t h e sex accessory glands. As noted
in t h e introductory remarks, i t seems likely
t h a t this is explained by t h e location of t h e
seminiferous tubules near t h e source of endogenous testosterone in t h e Leydig cells. Thus t h e
562
CHARLES J. FLICKINGER
seminiferous epithelium normally receives a
greater amount of testosterone t h a n most
other organs and i t s supply is not adequately
replaced by administration of testosterone sufficient to maintain other androgen-dependent
organs in a normal state (Heller et al., '70;
Walsh and Swerdloff, '73).
Alterations in testosterone e n a n t h a t e treated rats differed from ultrastructural
changes in t h e testis of men administered testosterone propionate (Barham and Berlin, '74)
with respect to alterations in spermatogonia
and spermatocytes. In both cases, however, decreased numbers of spermatids and changes in
those t h a t remained were reported, and Sertoli cells had similar features, including apparent uptake of degenerating germ cells. The
enclosure of degenerating cells by Sertoli cytoplasm, accompanied by a n increase in t h e size
and abundance of lipid droplets in t h e cytoplasm of t h e Sertoli cells, has been observed in
many other instances in which germ cells degenerate (for references see Flickinger,
'77a,c), and t h e lipid may be derived from t h e
breakdown of parts of t h e germ cells.
Epididymis
Some of t h e changes in t h e epididymis in
testosterone enanthate-treated rats, such a s
decreased epididymal weight and t h e progressive disappearance of sperm from t h e lumen of
most parts of the epididymis, can be accounted
for by t h e diminished production of sperm by
t h e testis. Similarly, changes in t h e contour of
t h e epithelium in t h e proximal part of t h e terminal segment may have resulted from collapse of the lumen in t h e absence of sperm.
The presence of very large amounts of cellular debris and degenerating sperm filling t h e
distal part of t h e terminal segment seem likely t o be due to t h e failure of sperm t o survive
in this region where they are normally stored.
Several possible explanations for this can be
suggested. First, t h e epididymis receives a
dual supply of androgen, both from t h e blood
and in t h e luminal fluid from t h e testis (White
and Hudson, '68; Waites and Setchell, '69;
Hanssori e t al., '75). Since t h e Leydig cells of
the testis were very severely suppressed, i t
seems likely t h a t t h e normal supply of androgen in t h e luminal fluid was not adequately
replaced by th*e administration of exogenous
testosterone, with possible deleterious effects
on t h e luminal sperm or t h e epididymal epithelium. Second, if sperm were not transported normally beyond t h e distal cauda epi-
didymis, they may have aged there, died, and
begun to disintegrate. Finally, under the influence of t h e treatment t h e germ cells t h a t
reached t h e cauda may have been immature
or defective. It is not possible to choose between these alternatives, but the first two
imply some changes in t h e interaction between sperm and epididymal epithelium in the
distal cauda epididymidis. Such a change
could contribute to t h e antifertility action of
testosterone enanthate by providing a n additional barrier t o t h e escape of normal sperm
from the male reproductive tract.
It is to be emphasized t h a t t h e degenerating
sperm and cellular debris present in t h e distal
cauda epididymidis of treated rats occupied
almost t h e entire lumen; no areas with normal
sperm were found. Cooper and Hamilton ('77)
have described t h e presence of some degenerating sperm in t h e distal epididymis and vas
deferens of normal mammals and have questioned t h e significance of reports of degenerating sperm in t h e cauda epididymidis of
rats receiving antifertility agents. However,
t h e difference between t h e normal and the
situation described here and in previous publications (Flickinger and Loving, '76; Flickinger, '77b,c) is dramatic. In t h e normal the
masses of degenerating sperm occupied only a
fraction of t h e lumen and t h e proportion of degenerating sperm remained small, but in
treated animals most of t h e lumen contained
debris and almost all t h e sperm appeared t o be
degenerating.
An interesting feature not previously observed in our studies of various antifertility
agents was t h e appearance of many apparently phagocytic cells in t h e epithelium of the
middle segment and proximal part of t h e terminal segment of testosterone enanthatetreated rats. The nature of these cells has not
definitely been determined, but they have
many features characteristic of monocytes,
including their nuclear morphology, large
Golgi apparatus, rough endoplasmic reticulum and dense cytoplasmic granules (Bloom
and Fawcett, '75). The presence of phagocytic
vacuoles makes i t seem unlikely t h a t the cells
represent lymphocytes, which are not normally phagocytic, even though other cytological
features may be consistent with those of the
unusual lymphocytes said t o occur in t h e r a t
(Hoffer et al., '73). The presence of phagocytosed material may make i t appropriate to
consider these cells as macrophages, which is
in accord with their aggregation in large
EFFECTS OF TESTOSTERONE
groups in the epididymal epithelium. Whatever their identity, the functional significance of phagocytic cells in the epididymal
epithelium of treated animals and the origin
of the ingested material remain uncertain.
The phagocytes were never observed in the
lumen of the epididymis. Phagocytes in the
epithelium might provide a means for disposal
of disintegrating germ cells located within the
intraepithelial cavities observed in some
specimens. However, no preferential association between the phagocytic cells and the epithelial cavities was detected.
Sex accessory glands
The sex accessory glands, represented by
the ventral prostate and the seminal vesicles,
were maintained a t close to normal weight,
and the ultrastructure of the epithelium suggested that normal secretory activities continued in the presence of testosterone enanthate. Other organs were not investigated,
but if other androgen-dependent organs and
male sexual characteristics fared as well as
the prostate and seminal vesicles it is likely
that male characteristics were generally well
maintained, even though spermatogenesis
was severely suppressed.
ACKNOWLEDGMENTS
The author is indebted to Miss Mary Stuart
for technical assistance and to Mr. Eric Sun
for injecting the animals. This research was
supported by a grant (1 R01 HD 10073) from
the National Institute of Child Health and
Human Development.
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Ewing, L. L., B. Schanbacher, C. Desjardins, and V. Chaffee
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CHARLES J. FLICKINGER
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PLATES
PLATE I
EXPLANATION OF FIGURES
1 Light micrograph of normal rat testis. The seminiferous tubules contain Sertoli
cells and germ cells in different stages of development. Present are spermatogonia
next to t h e basal lamina, spermatocytes (P),and many acrosome phase spermatids
(T). Leydig cells (L) lie in the interstitial tissue between the tubules. N, Sertoli
cell nuclei. x 260.
2 , 3 Light micrographs of the testis of a rat treated with testosterone enanthate for
12 weeks. In the seminiferous tubules, Sertoli cells, spermatogonia, spermatocytes
(P), and early spermatids (S) are present, but late spermatids are virtually absent. Degenerating germ cells are numerous and are identified by their dense
granular appearance. Many are apparently early spermatids (DS), while others
are primary spermatocytes (DP) as indicated by their size and location. Leydig
cells in the interstitial tissue are inconspicuous due to their small size. Figure 2
X 290; figure 3 X 250.
566
EFFECTS OF TESTOSTERONE
Charles J. Flickinger
f’LATE 1
567
EFFECTS OF TESTOSTERONE
Charles J Fhckrnger
PLATE 2
EXPLANATION OF FIGURES
568
4
Electron micrograph of a degenerating cell in the seminiferous epithelium of an animal treated for 8 weeks with testosterone enanthate. The cell is judged to be degenerating by its extreme density and the disruption of the normal architecture of its
organelles. I t is identified as a n early spermatid of the cap phase because of the persistence of an acrosomal cap (A). The degenerating cell is surrounded by Sertoli cell
cytoplasm (C). x 11,000.
5
Low power electron micrograph of the seminiferous epithelium in a rat administered testosterone enanthate for 8 weeks. Constituents of Sertoli cells include part
of the nucleus (N), a cytoplasmic lipid droplet (L), dense elongate inclusions (I), and
degenerating cellular material (D). Some normal appearing germ cells are represented, including a cap phase spermatid 6)and some preleptotene spermatocytes
(P). X 6,400.
EFFECTS OF TESTOSTERONE
Charles J. Flickinger
PLATE 3
569
PLATE 4
EXPLANATION OF FIGURES
6
Part of a Leydig cell of a normal rat. Much of t h e cytoplasm of the large cell is occupied by smooth endoplasmic reticulum (El and mitochondria (M). R, rough endoplasmic reticulum; N, nucleus. X 17,000.
7 Leydig cell from a rat treated for 8 weeks. Cytoplasmic organelles including smooth
endoplasmic reticulum (E) and mitochondria (M) are not abundant because the cell
is very small compared to normal (cf. fig. 6 ) . The nucleus (N) contains prominent
clumps of heterochromatin. Leydig cells are frequently dense in testes fixed with
the aldehyde mixture used in this study. X 17,000.
570
EFFECTS OF TESTOSTERONE
Charles J. Flickinger
PLATE 4
571
PLATE 5
EFFECTS OF TESTOSTERONE
Charles J Flickinger
EXPLANATION OF FIGURES
8
Light micrograph of the middle segment of the epididymis in a control rat. The epididymis is lined by a columnar epithelium of moderate height, and t h e lumen (L)
contains many sperm. X 220.
9 Middle segment in a rat administered testosterone enanthate for 12 weeks. Sperm
are virtually absent from the lumen (L) which contains only a few round cells.
Large cavities (C) are conspicuous within the epithelium. x 220.
10 Low power electron micrograph of the middle segment of t h e epididymis in a rat
treated for 12 weeks. A portion of an intra-epithelial cavity (C) is surrounded by
several principal cells. Microvilli project from the surface of the cells into the cavity, which contains a light granular material and a central mass of debris consist.
ing of small spherical objects and dense fibers. A portion of the epididymal lumen
(L) appears in the upper left hand corner of the field. Present within the epithelium is a dense polymorphous mass of material (D)surrounded by a thin rim of
pale-staining cytoplasm. X 4,500.
572
EFFECTS OF TESTOSTERONE
Charles J. Flickinger
PLATE 6
PLATE
I
EXPLANATION O F FIGURES
11, 12 Examples of the pale-staining cells that resemble leukocytes in t h e epithelium
of the middle segment in a rat administered testosterone enanthate for 12
weeks. Prominent in the cytoplasm are dense membrane-bound structures (D)
t h a t contain a variety of materials, including cellular organelles. N, nucleus. In
figure 11 note the large Golgi apparatus (GI and the cytocentrum (HI. Figure
11 X 15,000; figure 12 X 11,000.
5 74
EFFECTS OF TESTOSTERONE
Charles J. Flickinger
PLATE 7
PLATE 8
EXPLANATION OF FIGURE
13 Low magnification electron micrograph of a mass of pale-staining cells in the epithelium of the middle segment of a rat treated for 12 weeks. The cells have irregular interdigitated outlines, and their cytoplasm contains dense structures (D) with
a polymorphous content, which resemble lysosomes. X 8,500.
576
EFFECTS OF TESTOSTERONE
Charles J Flickinger
PLATE 8
577
PLATE 9
EXPLANATION OF FIGURES
14 Light micrograph of the proximal part of the terminal segment of a control rat.
The lumen (L) contains many sperm and is lined by a columnar epithelium. X 285.
15 Proximal part of the terminal segment in a rat treated for 12 weeks. Sperm are ab-
sent from t h e small lumen (L). The epithelium is tall and has an irregular undulating outline, perhaps as a result of collapse of the lumen. Some vacuolated light
cells with dense cytoplasmic granules are evident (arrow). X 285.
16 Light micrograph of the distal part of the terminal segment in a control rat. The
large lumen (L) is surrounded by a low columnar epithelium and a smooth muscle
coat (M). x 285.
17 Distal part of the terminal segment of a r a t treated for 12 weeks. The epithelium
is a low columnar type as in the normal, but the luminal contents are altered.
Among clumps of sperm (S) are round cells, spherical masses of cytoplasm (0,and
debris. X 285.
578
PLATE 9
EFFECTS OF TESTOSTERONE
Charles J. Flickinger
579
PLATE 10
EXPLANATION OF FIGURES
18 Lumen of the distal part of t h e terminal segment in a rat treated for 12 weeks. The
luminal material includes spherical masses of cytoplasm (C) some of which contain
a nucleus (N), a few sperm (S), a flocculent material (F), and scattered debris.
X 4,000.
19 Light micrograph of seminal vesicle of a r a t treated with testosterone enanthate
for 12 weeks. The epithelium (El contains secretory granules visible as dense spots
surrounded by a clear rim. Secretory material (A) is present in the lumen between
the folds of epithelium. X 230.
20
580
Light micrograph of ventral prostate of a rat treated with testosterone enanthate
for 12 weeks. The prostatic acini are lined by a medium to tall columnar epithelium (E). X 230.
EFFECTS OF TESTOSTERONE
Charles J. Flickinger
PLATE 10
581
PLATE 11
EXPLANATION OF FIGURES
582
21
Seminal vesicle of a rat treated for 12 weeks. The epithelial cells contain abundant
organelles associated with the formation of secretions: rough endoplasmic reticulum (R), Golgi apparatus (GI, and t h e characteristic secretory vacuoles (S) that
contain a dense secretory granule surrounded by a light rim. L, lumen of the gland.
X 15,500.
22
Ventral prostate of a rat treated for 12 weeks. The prostatic epithelial cells display
normal organelles, including abundant rough endoplasmic reticulum (R),t h e Golgi
x 9,000.
apparatus, and secretory vacuoles 6).
EFFECTS OF TESTOSTERONE
Charles J . Flickinger
PLATE 11
583