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The influence of progestin and androgen on the fine structure of the male reproductive tract of the rat. I. General effects and observations on the testis

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The lnfluence of Progestin and Androgen on the Fine
Structure of the Male Reproductive Tract of the Rat
I. GENERAL EFFECTS AND OBSERVATIONS ON THE TESTIS
CHARLES J. FLICKINGER
D e p a r t m e n t of Anntomy, School of Me dic ine , Uniuersitg of Vir ginin,
Cknrlottesuille, Virginia 22901
ABSTRACT
The combination of a progestin and androgen has received attention a s a possible male contraceptive. The progestin is thought to reduce
gonadotropin release and suppress spermatogenesis, while the sex accessory
organs and male characteris tics are maintained by the simultaneous administration of testosterone. In the present study, the histology and ultrastructure of
parts of the male reproductive tract of rats treated with medroxyprogesterone
(Provera, Upjohn) (1 mg/100 g body weight/day) alone and combined with testosterone (15, 30, o r 100 pg1100 g/day) were studied following treatment for
up to 16 weeks. The testes and epididymides of rats administered Provera alone
or Provera and testosterone weighed less than those of control rats. The weights
of the accessory glands of rats treated with Provera were greatly reduced; it was
possible to maintain them at approximately control levels by simultaneously
administering sufficient testosterone (100 pg/100 g body weight/day). The fertility of some of the animals was tested by caging them with female rats, and
none of the treated rats tested i n this way was fertile.
Similar microscopic alterations were present in the testes of animals administered Provera alone or Provera and different levels of testosterone. Spermatogonia, spermatocytes, and early spermatids were abundant in treated rats and
did not show ultrastructural changes. However, many degenerating or necrotic
spermatids of the cap phase (approximately stages 6-7) and later were present.
Late spermatids of the acrosome and maturation phases were rare. Some necrotic
spermatids were surrounded by Sertoli cells, and parts of spermatids lay within
lysosome-like structures in the cytoplasm of Sertoli cells. Many large lipid droplets were also present in Sertoli cells of treated rats. Leydig cells were smaller
i n treated animals than in control rats. The results suggest that germ cells can
develop up to cap phase spermatids but then undergo degeneration. These alterations in spermatogenesis may be responsible in large part for the antifertility
effect of the progestin and androgen combination.
Some rats were permitted to recover following the end of treatment. The microscopic appearance of the testis returned to normal within three to six weeks,
although epididymal alterations persisted in some animals six weeks after the
end of treatment. By 9 to 12 weeks after the end of treatment the reproductive
organs had a normal microscopic appearance in all the rats studied.
Many different kinds of compounds have
been studied for their antifertility effects
in the hope that a suitable chemical contraceptive for men can be found. Recently
the combination of a progestin and an androgen has been tested and is believed to
show considerable promise as a male contraceptive (Segal, '73), because it is reported to produce reversible infertility while
libido and male sex characteristics are
maintained (Frick, '73; Coutinho and Melo,
ANAT. REC., 1 8 7 : 4 0 5 4 3 0 .
'73). The rationale behind the administration of a progestin-androgen combination
for male contraception is as follows (Terner
and MacLaughlin, '73; Kragt et al., '73).
The progestin is thought to diminish gonadotropin release and suppress spermatogenesis. However, the Leydig cells of the
cells of the testis are also suppressed, resulting in decreased secretion of testosterReceived Mar. 18, '76. Accepted Oct. 1 , '76.
405
406
CHARLES J . FLICKINGER
one. Secondary sex characteristics and libido are then maintained by the simultaneous administration of exogenous testosterone.
Stumes in the rat have shown that it is
indeed feasible with this combination to
abolish fertility and to maintain the weight
of the sex accessory glands and sexual behavior (Terner and MacLaughlin, '73;
Kragt et al., '73). The influence of this
regimen on the structure of the male reproductive tract has not previously been
studied, however, and many questions remain concerning its effects on the reproductive organs. Therefore, in the present
investigation, the influence of a progestin
alone and in combination with testosterone
on the histology and ultrastructure of the
testis, epididymis, and sex accessory glands
of the male rat were studied at intervals
up to 16 weeks of treatment. The general
aim of the study was to determine the effects of these agents on the structure of
the male reproductive tract as a basis for
understanding the antifertility action of
these compounds. An important characteristic of a potential male contraceptive
agent is that its action be reversible. Consequently, observations were also made on
the weight and microscopic appearance of
the reproductive organs of rats permitted
to recover following the cessation of treatment with progestin and testosterone.
The present article is concerned with
the general effects of the progestin-androgen regimen, including the influence of
the agents on the weights of the reproductive organs and on fertility, and with the
histology and ultrastructure of the testis.
Specific objectives with respect to the study
of the testis were to determine which
stages of spermatogenesis are affected by
the treatment, and the nature of possible
cytological changes in germ cells, Sertoli
cells,and Leydig cells. The companion paper will consider alterations in different
parts of the epididymis and in the prostate
and seminal vesicle (Flickinger, '77).
MATERIALS A N D METHODS
Young adult male rats of the SpragueDawley strain weighing approximately 200
g were obtained from Hilltop Lab Animals,
Inc., Scottdale, Pennsylvania. A total of 55
animals was used; of these, 17 were treated
with a progestin alone, 23 were administered progestin and testosterone combined,
and 15 served as controls. Animals were
maintained at 20-22°C and had constant
access to food and water.
Rats were injected subcutaneously with
steroids in 0.1-0.2 ml of sesame oil. Control animals received an equivalent amount
of the oil alone. The compounds and the
doses administered in the different experiments are listed below.
Experiment I . (table 1). Progesterone
was administered at a dose of 2 mglratl
TABLE 1
The f i n a l w e i g h t s of the reproductiue organs arid t h e f i n a l b o d y w e i g h t s of rats treated w i t h progesterone
Testis
Time
iwk )
1
2
4
Seminal
ve sic Le
Prost ate
Body
weight
(average)
~
Treatment
(Expt. No. 1)
Control (2)
Prog 2 rng (1)
Prog 1 0 m g (1)
Control (2)
Prog 2 mg (1)
Prog 10 mg (1)
Control (2)
Prog 2 mg (1)
Prog 10 mg (1)
7-8
Epididymis
Control (1)
Prog 10 m g (2)
g/100 g
9%
control
g/100 g
0.57
-
0.14
0.63
0.46
0.44
0.45
0.42
0.38
0.38
0.33
0.33
111
0.43
75
96
98
90
90
100
8
70
0.10
0.14
0.12
0.11
0.1 1
0.13
0.12
0.10
0.14
0.10
%
control
g/lOO g
control
g/100 g
control
g
-
0.13
0.08
-
0.13
0.10
0.08
0.10
0.05
0.09
-
255
315
230
303
340
278
38 8
330
350
485
450
71
100
92
92
0.09
62
69
0.12
0.08
0.11
67
92
-
-
0.15
-
92
77
0.09
0.07
0.16
0.05
60
47
71
-
31
0.13
0.04
0.05
0.15
0.05
77
62
-
50
90
31
38
33
The number of animals is shown i n parentheses after the designation for the treatment. The average values for the testis.
epididymis, and seminal vesicle include the two organs from each rat. The number of rats is smaller than i n the following
experiments, since this w a s a preliminary experiment to test the effects of progesterone on the reproductive organs. The organ
weights are shown in g per 100 g body weight and as 8 control. A b b r e ~ ~ i a t i o nProg
s:
2 mg, 2 mg progesteronelratlday, Prog
10 mg, 10 mg progesteroneiratlday.
PROGESTIN AND ANDROGEN. I
day, or 10 mglratlday. Rats were killed a t
intervals of 1 , 2, 4, 7, and 8 weeks. In this
preliminary experiment, for simplicity a
constant dose of progesterone was used.
In all the subsequent experiments the dose
of steroids was adjusted to the weight of
the individual animal. The lower dose of
2 mglratlday was selected because it had
been reported to alter spermatogenesis in
rats (Kar et al., '67). When this effect was
not observed i n our animals, the dose of
progesterone was increased to 10 mg/rat/
day in a n effort to influence spermatogenesis.
Experiment 2. (table 2). Medroxyprogesterone (Provera, Upjohn) was administered at a dose of 1 mg/100 g body weight/
day. Rats were killed a t intervals of 4, 8,
and 16 weeks. This dose of Provera was
used because i t is similar to that employed
in previous studies on the effects of ProV e r a on the reproductive tract of male rats
(Terner and MacLaughlin, '73). This
amount of Provera resulted in microscopic
alterations in the reproductive tract, and
therefore it was continued in the following
experiments, i n which the amount of testosterone was varied.
Experiment 3 . (table 2). Provera 1 mg/
100 g/day was given in combination with
testosterone propionate 15 pg/lOO g/day.
Animals were killed a t intervals of 4, 8,
and 16 weeks. This amount of testosterone
was used because a similar dose had been
reported to prevent or diminish the effects
of various progestins (Terner and MacLaughlin, '73).
Experiment 4. (table 2). Provera 1 mg/
100 g/day plus testosterone propionate 30
pgl100 glday were given. Rats were killed
at intervals of 4, 8, and 16 weeks. This
dose of testosterone was employed because
the weight of the sex accessory glands was
not maintained a t control levels i n the
presence of 15 pg/lOO g/day. Therefore,
the dose of testosterone was doubled.
Experiment 5 . (table 2). Provera 1 mg/
100 glday plus testosterone propionate 100
pg/lOO g/day were administered. Animals
were killed at intervals of four and eight
weeks. Since the weight of the sex accessory glands remained below control levels
in the presence of testosterone 30 pg/100
glday, the dose of testosterone was increased. The level of 100 pg1100 glday was
407
used because it was reported that this was
sufficient to maintain the sex accessory
glands of castrated rats (Ramirez and McCann, '65).
Some control and treated animals were
not killed during the course of the experiment, but instead were permitted to recover for 3, 6, or 9 weeks after the cessation of treatment as shown i n table 3.
Medroxyprogesterone (Provera) was obtained through the courtesy of Millard W.
Beucler and Marvin R. Guthaus of the
Upjohn Co., Kalamazoo, Michigan. The
progesterone and testosterone propionate
were purchased from the Sigma Chemical
Co.,St. Louis.
The reproductive organs, including testes, epididymides, ventral prostate, and
seminal vesicles were removed and weighed.
Whole testes were immersed for one hour
in a glutaraldehyde, formaldehyde, and
picric acid fixative (It0 and Karnovsky,
'68), 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
the outside of the testes, diced, and fixed
for a n additional one hour. After fixation
for a total of two hours i n aldehyde, the
tissue blocks were rinsed with 0.1 M cacodylate buffer and post-fixed for one hour
in 1% OsO4 in 0.1 M cacodylate buffer a t
pH 7.3. The tissue was dehydrated in a
graded series of ethanols followed by propylene oxide, and was embedded i n Araldite.
Sections 1 pm thick for light microscopy were cut with glass knives, mounted
on slides, and stained with 0.5% toluidine
blue in 0.5 % sodium borate. Thin sections
showing silver to pale gold interference
colors were cut with a diamond knife,
mounted on uncoated copper grids, and
stained with lead citrate (Reynolds, '63).
The preparations were examined and photographed using a Philips EM-300 electron
microscope.
The sizes of profiles of Leydig cells of
the testis in sections for light microscopy
at the 8-week interval were estimated in
the following way. Regions of accumulation of interstitial tissue between three or
more seminiferous tubules were chosen at
random for study. Within these regions,
the length and width of all the cells iden-
4
3
2
15pg
P v f T 30pg
v
h + T
Control
Pv+TlOOpg
4
5
+
h.
Pv+T 15pg
Pv+T 30pg
Pv T 100 p g
2
3
P
Treatment
Control
Control
Pv
P v + T 15pg
Pv+T 30pg
2
3
4
5
Expt.
No.
0.42
0.16
0.18
0.18
0.25
0.38
0.16
0.16
0.16
0.19
0.36
0.13
0.18
0.16
g/100 g
36
50
44
-
42
42
42
50
-
38
43
43
60
-
% control
Testis
0.03
0.04
0.05
0.08
0.13
0.04
0.05
0.04
0.08
0.14
0.04
0.06
0.05
o.ii
g/100 B
29
43
36
-
31
38
31
62
-
27
36
45
73
-
5% control
Epididymis
0.11
0.02
0.05
0.07
0.14
0.14
0.02
0.06
0.06
0.14
0.13
0.03
0.05
0.04
g/100 g
23
38
31
-
14
43
43
00
-
0.01
0.04
0.04
0.06
0.08
0.02
0.04
0.05
0.12
0.07
0.03
0.04
0.06
0 07
18
45
64
27
g/100 g
-
43
57
86
-
25
50
63
150
-
14
57
57
86
-
% control
Seminal
vesicle
% control
Prostate
323
363
328
455
353
345
335
383
52 1
42 1
473
415
41
0 .
._
45 1
g
body weight
(average)
Each value is the average of the individual organ weights from two rats, except for F'v 16 weeks which is the average from three animals. The values for
the testis, epididymis, and seminal vesicles include the two organs from each rat. The weights are shown in g per 100 g hody weight and in % control.
Abbreviations: Pv, Rovera 1 mg/100 g/day; P v + T, Provera 1 mg/100g/day plus testosterone 15, 30, or 100 f i g / l O O g/day a s indicated.
16
8
4
Time
(wk)
The weights of the reproductive organs and t h e f i n a l body weights of rats treated w i t h Prouera or w i t h Provera
combined w i t h testosterone for 4, 8, or 1 6 weeks
TABLE 2
??
9
m
0
z
EE
9
CI
k
25
409
PROGESTIN AND ANDROGEN. I
TABLE 3
The t y p e a n d l e n g t h of t r e a t m e n t a n d t h e mlcroscop?c nppearunce of rats
v e r m t t t e d t o recover a f t e r t h e e n d of a t r e a t m e n t p e n o d
Time of
recovery
(wk)
Expt. No.
Length of
treatment
(wk)
Treatment
3
3
3
6.5
6
Control
Pv
Pv+T 15pg
16
16
16
16
16
7
7
Pv+T 30pg
P v + T 30pg
16
16
8
9
Pv
Control
T 100 p g
8
8
Pv+T 15pg
Pv
9
+
Pv + T 100 p g
12
P v + T 15pg
+ indicates a normal microscopic appearance, while
tifiable as Leydig cells were measured using a Zeiss optical micrometer. Assuming
the profiles of the cells to be eliptical, the
area of a cell represented within a section
was estimated by multiplying the length/
2 X width/2 X T. Twenty-five cells were
measured for each of four rats treated with
Provera (Experiment 2), two rats treated
with Provera and testosterone (Experiment
5), two normal untreated animals, and one
control rat. The mean areas of Leydig cells
in sections of control and normal rats were
compared with those of treated animals
using a T test.
The fertility of some of the rats was
tested by caging each male rat for five days
with two female rats and examining the
females one week later for the presence
of fetuses. Animals tested in this way included seven controls, four rats treated
with Provera (Experiment 2) and eight
animals treated with Provera and testosterone (Experiments 3 , 5).
RESULTS
General observations
Progestin
A preliminary experiment (Experiment
1) was carried out using progesterone as
the prototype and representative of the
class of progestational agents. Rats received 2 mg or 10 mg of progesterone daily
for up to eight weeks. A dose similar to the
lower one had been reported to arrest sper-
8
16
Microscopic
appearance
T
-
+
+
testes
rest
-
+
+
+
testes
rest
-
+
+
+
- designates the persistence of alterations
matogenesis in male rats (Kar et al., '67).
Although the weight of the prostate and
seminal vesicle of treated animals was in
most cases 30-50% that of control animals
after treatment for four weeks or longer
(table l), alterations in the height of the
epithelium and the cytology of epithelial
cells in the prostate and seminal vesicles
were minimal, and the histology of the
testis and epididymis appeared unchanged.
Therefore, we turned to medroxyprogesterone (Provera), because it is a more potent
progestational agent and was used in
studies on the effects of progestin-testosterone combinations in rats by others
(Terner and MacLaughlin, '73). Treatment
with Provera 1 mg/lOO g/day (Experiment
2) resulted in a decline in the weight of
the prostate and seminal vesicles in most
animals to 10-25% control values after
treatment for four weeks (table 2). The
weight of the testis and epididymis of
treated animals decreased to about 3040 % the control.
Progestin and testosterone combined
In our first experiment in which testosterone was given along with Provera (Experiment 3), rats were administered 15 cLg
testosterone/lOO g/day in combination with
1 mg Provera/100 g body weightlday, since
it had been reported (Terner and MacLaughlin, '73) that even a small dose of
testosterone such as this was capable of
preventing or decreasing the effects of pro-
410
CHARLES J. FLICKINGER
gestins. However, we found that although
the prostate and seminal vesicles of these
rats were larger and weighed more than
in rats administered Provera alone, they
weighed only about one-half those of control animals (table 2). Therefore, in the
next experiment (Experiment 4), we maintained the amount of Provera a t 1 mg/100
glday and increased the accompanying
testosterone to 30 pg/lOO g/day. However,
the weights of the prostate and seminal
vesicles of treated animals still did not
equal those of controls (table 1). Thus, in
the final experiment (Experiment 5), we
utilized Provera plus 100 pg testosterone/
100 g/day. At this level, the weights of the
prostate and seminal vesicles of treated
animals approximated or exceeded those
of control rats. The testes and epididymides
of animals treated with Provera and the
different doses of testosterone (Experiments
3 , 4, 5) were small, and in the majority of
treated rats weighed approximately half
those of control animals.
Fertility
The fertility of some of the treated rats
was tested by caging them with two female
rats. Since our main interest was in the
histology and ultrastructure of the reproductive organs, the number of animals
tested in this way is small, but the results
are nevertheless of interest in relation to
the morphological observations. All but
one of the seven controls impregnated a t
least one female. In contrast, none of the
four rats treated with Provera for eight
weeks (Experiment 2) or the eight animals
treated with Provera and testosterone for
eight weeks (Experiments 3 , 5) impregnated a female. It is uncertain precisely
how many of the rats administered Provera
and testosterone actually mated, but mating did occur i n a t least some instances
since vaginal plugs were observed in females placed with two of the treated males.
Since most of our animals were killed for
microscopic study during the course of the
treatment, we have little data on the recovery of fertility after the end of treatment, except that one of two animals tested
eight weeks after the end of treatment
with Provera and testosterone (Experiment
5) had regained fertility.
Testis
The structure of the testis of normal rats
resembles that of other mammals (Burgos
et al., '70). Seminiferous tubules contain
Sertoli cells and combinations of germ cells
in different stages of development (fig. 1).
The interstitial tissue includes testosterone-secreting Leydig cells, blood vessels,
lymphatics, and other connective tissue
elements (Fawcett et al., '73). The testes
of control rats administered the oil vehicle
did not differ from the normal.
Alterations in the testes of rats treated
with Provera were observed at all the intervals studied, including the earliest samples which were taken after treatment for
four weeks. Changes in the microscopic
structure of the testes of rats treated with
Provera and testosterone closely resembled
those observed after treatment with ProVera alone. Furthermore, the structure of
the testis did not vary significantly with the
amount of testosterone added to the ProVera. Therefore, the microscopic structure
of the testes of animals treated with ProVera alone and with Provera and the different levels of testosterone will be described together. Since no consistent progression of the alterations after four weeks
of treatment was observed, to reduce redundancy the different intervals are not
all sequentially illustrated and described
individually.
At the light microscope level, spermatogonia, primary spermatocytes, and early
spermatids up to the cap phase were readily
visible, but later spermatids were rare or
appeared to be completely absent i n the
light microscope preparations (figs. 2, 3).
The testes of treated animals showed
numerous large lipid droplets and denselystaining cells that appeared to be degenerating within the seminiferous epithelium. In different profiles of the seminiferous tubules, different types of the early
germ cells were present. It was thought
that this might reflect the presence of different stages of the cycle of the seminiferous epithelium from which late spermatids
were absent or depleted in number, but a
systematic comparison of each of the stages
of the cycle was not carried out.
Study of the seminiferous epithelium
with the electron microscope revealed no
morphological alterations in spermatogonia,
spermatocytes, or Golgi phase spermatids
(figs. 4-7). Spermatogonia were recognized
as round or oval cells close to or i n contact
with the base of the seminiferous epithelium
PROGESTIN AND ANDROGEN. I
(fig. 5 ) . The cytoplasm contained clusters
of free ribosomes but only small amounts
of membranous organelles such as endoplasmic reticulum and Golgi apparatus.
The round to oval nucleus had a uniform
distribution of chromatin in some cells
which may represent type A spermatogonia.
Other nuclei that had more prominent
clumps of chromatin may be those of type
B spermatogonia. Many primary spermatocytes were present (fig. 4). These were
larger than spermatogonia and in favorable
sections their cytoplasm could be seen to
contain a larger Golgi apparatus and more
abundant profiles of endoplasmic reticulum. The round nuclei of the primary spermatocytes presented a variety of chromatin
patterns and nucleolar morphologies, probably reflecting different stages of meiotic
prophase. The identification of many synaptinemal complexes (fig. 4) suggested
that pairing of homologous chromosomes
occurred and that meiosis proceeded
through zygotene i n treated animals. Golgi
phase spermatids were present (fig. 6).
Their most prominent features included
the usual round nucleus, a large Golgi apparatus, proacrosomal granules, and the
single large acrosomal granule, which was
enclosed i n the acrosomal vacuole and apposed to one side of the nucleus (fig. 6).
Some normal-appearing spermatids of the
early cap phase were present, but as described in more detail below other cap
phase spermatids were degenerating. Those
of normal appearance (fig. 7) displayed a
large Golgi apparatus and the characteristic acrosomal vacuole, which formed a
cap over the anterior surface of the round
spermatid nucleus. Additional features of
these cells included mitochondria located
around the margin of the cytoplasm, cytoplasmic vesicles, and, frequently, a chromatoid body. Intercellular bridges were
observed between spermatocytes and between spermatids. Although the early spermatogenic stages of spermatogonia, spermatocytes, and early spermatids remained
abundant in sections, i t should be noted
that changes in their numbers were not
ruled out, because a quantitative analysis
of the different types was not undertaken.
As suggested by the light microscope observations, degenerating cells were numerous i n samples of the testes of treated
animals viewed with the electron microscope (fig. 8). These cells displayed in-
411
creased cytoplasmic density, organelles
with disrupted membranes, and myelin
figures. The severity of the morphological
changes suggests that many of the cells
were irreversibly damaged and thus may
be considered necrotic (Robbins, '74). Many
of these cells were identified further as
early spermatids i n the latter part of the
cap phase (approximately stages 6-7: Leblond and Clermont, '52) because they
contained the acrosomal cap that is characteristic of these cells. The presence of
some degenerating cells has been described
in normal testes (Oakberg, '56; Clermont
and Bustos-Obregon, '68), but spermatogonia and spermatocytes are involved. Degenerating spermatids such as those seen
in treated animals were not observed in
our samples of normal and control rats.
The morphological changes that precede
those obvious necrotic alterations were not
identified with certainty. However, some
spermatids displayed increased cytoplasmic
density, numerous cytoplasmic vacuoles,
and irregularly shaped nuclei, and these
features may reflect early degenerative
changes.
Few later spermatids of the acrosome
and maturation stages remained in the
seminiferous epithelium, but some could
usually be identified with the electron microscope. Many of those present, however,
appeared also to be undergoing degeneration and death as indicated by the presence of dense cytoplasm and alterations
in the architecture of the acrosome, tail
fibers, and other organelles (fig. 9).
Sertoli cells in treated rats retained
many normal features (fig. 4). They had
an irregular shape, with the nucleus and
perikaryal cytoplasm being located near
the basal lamina, and with long processes
extending throughout the seminiferous
epithelium in the interstices between the
varieties of germ cells. The nuclei of Sertoli cells contained much euchromatin and
a large nucleolus. Their cytoplasm displayed
multiple stacks of Golgi cisternae, scattered rough endoplasmic reticulum, numerous tubules and vesicles of smooth endoplasmic reticulum, and mitochondria.
In the long processes of Sertoli cells, microtubules were abundant. The characteristic
junctional specializations between Sertoli
cells were conspicuous in treated rats. As
previously described (Flickinger and Fawcett, '67), they consisted of cisternae of
412
CHARLES J. FLICKINGER
endoplasmic reticulum lying parallel to the
plasma membrane and bundles of fine
filaments interposed between the endoplasmic reticulum and the cell surface. Since
late spermatids were reduced in number,
the junctional specializations of Sertoli
cells next to these germinal cells were uncommon. When present, however, they had
the normal morphology reminiscent of onehalf of one of the Sertoli-Sertoli specializations (Flickinger and Fawcett, '67).
Many large lipid droplets were readily
visible in the cytoplasm of Sertoli cells (fig.
10). In contrast to the lipid droplets of normal Sertoli cells which are prominent at
stage IX of the cycle of the seminiferous
epithelium (Ken- and DeKretser, '75), in
treated rats the lipid droplets were numerous in almost all of the profiles of seminiferous tubules. In addition, they frequently
were so large in treated animals as to indent the nucleus, and sometimes they appeared to displace other organelles. Although most of the lipid droplets in the
seminiferous epithelium were found in the
basal cytoplasm of Sertoli cells, a few germ
cells contained large lipid droplets as well.
The cytoplasm of Sertoli cells also contained numerous lysosome-like structures
with a polymorphous content of cellular
debris (figs. 8, 10). Parts of spermatids
were found in these bodies and necrotic
cells were surrounded by the cytoplasm of
Sertoli cells (fig. 8), suggesting that spermatids were phagocytosed and digested by
the Sertoli cells.
The Leydig cells of the interstitial tissue
appeared smaller in treated rats than in
controls at all the intervals studied. They
were not measured at all the intervals, but
this was done for the 8-week specimens in
an attempt to confirm the impression gained
from inspection of sections that the size of
Leydig cells in treated animals was reduced.
The mean area of a Leydig cell represented
in sections of normal animals was 96 f i 2
and of the control, 91 p2. For animals
treated with Provera for eight weeks (Experiment 2), the corresponding value was
only 24 p 2 , and in rats administered ProVera and testosterone (Experiment 5, testosterone 100 Wg/lOO g/day) it was 25 p2.
In both experiments, the difference between the treated and the control and between the treated and the normal is highly
significant (P < 0.001). Values similar to
those found here for normal and control
rats were also obtained for Leydig cells
measured in a larger number of control
animals in a previous study (Flickinger
and Loving, '76). Large amounts of smooth
endoplasmic reticulum are characteristic
of the ultrastructure of normal Leydig
cells. In treated animals, smooth tubules
and other normal-appearing organelles remained in the cytoplasm of Leydig cells,
but as indicated by the measurements performed with the light microscope, the
amount of cytoplasm was less than in normal or control rats.
Recovery
Rats allowed to recover following treatment with Provera or with Provera plus
testosterone for 8 or 16 weeks were killed
at intervals of up to 12 weeks after the
end of the treatment (table 3). Alterations
resembling those of treated animals persisted in the structure of the testis and
epididymis of two rats killed three weeks
after the end of treatment. By six weeks
after the end of treatment, the reproductive organs of two of four animals had a
normal histology and ultrastructure. In
the other two recovery appeared to have
begun with a return of abundant late spermatids in the testis, but the epididymis
continued to show alterations. Three animals killed 9 or 12 weeks after cessation
of treatment had a normal microscopic
structure, including a return of late stages
of germ cells in all the seminiferous tubules (fig. 11). As shown in the companion
paper (Flickinger, '77) the structure of the
epididymis of animals permitted to recover
for 9 to 12 weeks also returned to normal.
DISCUSSION
The observation of great diminution or
absence of late spermatids of the acrosome
and maturation stages, along with the
presence of degenerating spermatids of the
cap phase and beyond, suggests that in
the testes of rats treated with Provera or
Provera and testosterone germ cells develop approximately up to the early spermatid stages 6-7 and then undergo degeneration. In accord with this hypothesis,
there was a general lack of morphological
alteration in the less mature germ cells,
including spermatogonia and spermatocytes. However, as pointed out above,
PROGESTIN AND ANDROGEN. I
changes in the numbers of earlier stages
of germ cells might have gone undetected
since the morphological methods employed
in this study did not include a quantitative
analysis of the different cell types. In addition, it was not possible to identify with
certainty the stage of all the necrotic cells,
and it is conceivable that some of these
might have represented earlier germ cells.
In any event, the scarcity of late germ
cells helps to account for the small size of
the testes and their reduced weight in
treated rats. Furthermore, the alterations
in the development of spermatids seemingly
were of sufficient severity to be responsible
in large measure for the infertility of the
treated animals.
The mechanism by which Provera and
testosterone produce alterations in spermatogenesis is not definitely known, but
some speculations can be based on the
present observations. Provera could suppress spermatogenesis by reducing the release of gonadotropins. Although hormone
levels were not measured directly, the reduced size of Leydig cells in treated animals seems likely to be the result of decreased stimulation by gonadotropins. In
addition, the suggestion that gonadotropins
were reduced is supported by the similarity
between the testicular changes in the present study and those after hypophysectomy
(Clermont and Morgentaler, '55). Spermatids from stage 8 on were found to degenerate after hypophysectomy, while the
slightly earlier stages 6-7 appeared to be
the first to degenerate in the present study.
In the case of Provera administered alone,
diminution in LH levels and consequently
in testosterone secretion by the Leydig cells
would be expected adversely to affect spermatogenesis, since androgen binds to a
cytoplasmic receptor protein (CR) in Sertoli cells or early germ cells (Hansson et
al., '74, '75) and is necessary for spermatogenesis to proceed (Steinberger, '71). Although the role of FSH in maintaining
spermatogenesis has been debated, FSH
does appear to influence the seminiferous
tubules (Lostroh, '63; Hall, '70), interacting with Sertoli cells and/or early germ
cells to influence their synthetic capacities
(Means, '75) and specifically stimulating
the synthesis of androgen binding protein
(ABP) by Sertoli cells (Hansson et al., '74,
'75). Thus a reduction in FSH and conse-
413
quently in ABP synthesis might also affect
the seminiferous tubules. The observation
that the later stages of spermatogenesis
were affected in the present study is of
interest with regard to the contention that
FSH is necessary for maturation of spermatids beyond stage 15 in the rat (Steinberger, '71; Steinberger and Duckett, '67).
Changes in FSH and ABP might help to
account for the observation that changes
in spermatogenesis persisted in the presence of sufficient exogenous testosterone
to maintain the sex accessory glands. It is
likely that the seminiferous tubules in ProVera and testosterone-treated rats received
less than their normal supply of testosterone, despite the administration of exogenous testosterone sufficient to maintain
the sex accessory glands, because the tubules normally receive a greater supply of
androgen as a consequence of their proximity to the Leydig cells and because testosterone is accumulated in the seminiferous tubules by ABP (Hansson et al., '74).
Thus, if ABP were reduced as a result of
diminished FSH, accumulation of testosterone in the tubules would be diminished.
Then less testosterone would be available
for subsequent binding to the cytoplasmic
receptor protein (CR) of Sertoli cells or
early germ cells, which is thought to mediate the response of spermatogenesis to androgen (Hansson et al., '74).
Previous studies on the influence of progestational agents on the testis have
yielded varying results. Progestins, including progesterone and a variety of other
agents, have been reported to suppress
spermatogenesis in several species (Setty
and Kar, '67; Kar et al., '67; Patanelli and
Nelson, '59; Ericsson et al., '64; Skinner
and Adams, '69; Ericsson and Dutt, '65;
Skinner and Rowson, '69; Heller et al.,
'58, '59; MacLeod, '65), although little
influence on spermatogenesis has been reported with some compounds (Jackson, '69;
Steinberger, '71). A finding similar to the
present observations was that spermatogenesis in men proceeded no further than
early spermatids in the presence of norethandrolone (Heller and Clermont, '64). In
rats administered progesterone and other
progestins percutaneously , spermatogenesis was reported to be arrested at the secondary spermatocyte stage (Setty and Kar,
'67).
4 14
CHARLES J. FLICKINGER
The fate of at least some of the degenerating and necrotic spermatids appeared
to be phagocytosis by Sertoli cells, since
lysosome-like structures in Sertoli cells were
numerous and contained cellular debris
that included identifiable spermatid structures. In some cases, entire necrotic spermatids appeared to lie within Sertoli cells.
The suggestion that Sertoli cells take up
degenerating spermatids is in accord with
the activities of Sertoli cells in phagocytosis of injected particulates (Clegg and
MacMillan, '65) and damaged germ cells
under a number of deleterious conditions
(Lacy and Lofts, '65; Villar et al., '67; Hugon and Borgers, '66; Fkddy and Svoboda,
'67; Roosen-Runge and Leik, '68; Black,
'71) and their retention of residual bodies
(Smith and Lacy, '69; Brokelmann, '63;
Dietert, '66). It is not known if phagocytosis by Sertoli cells is the fate of all the
spermatids. Some altered germ cells may
also have been shed from the seminiferous
epithelium and transported to the epididymis to accumulate in the distal part of the
cauda epididymidis (see the companion
paper, Flickinger, '77).
The many lipid droplets in Sertoli cells
of treated animals could be the result of
accumulation of lipid from ingested germ
cells, as may be the case in the accumulation of lipid from ingested residual cytoplasm of spermatids at certain stages of
the cycle of the normal seminiferous epithelium (Niemi and Kormano, '65; Kerr
and DeKretser, '75). The lipid accumulation might also be due to an alteration in
the metabolism of the Sertoli cells themselves, since lipid accumulation is seen in
a variety of cells under pathological conditions (Robbins, '74). In any event, an
increase in lipid in Sertoli cells has been
observed in other cases in which spermatogenesis is disrupted (Lacy, '62; Lacy and
Lofts, '65; Collins and Lacy, '69; Chung,
'74).
The weight of the reproductive organs of
rats treated with Provera was less than
that of control animals at all the intervals
studied. In rats administered testosterone
along with the Provera, the weight of the
testes and epididymides remained less than
those of control animals, as might be anticipated since spermatogenesis remained
suppressed. It proved possible to maintain
the weight of the ventral prostate and
seminal vesicles at a level comparable to
controls by the addition of testosterone to
Provera, but the dose of testosterone required was greater than initially anticipated. The matter of maintenance of the
accessory glands in the presence of ProVera and different amounts of testosterone
will be considered further in the companion paper, which deals with the sex accessory glands (Flickinger, '77).
The number of animals allowed to recover in the present study is relatively small
compared to those killed during the course
of treatment, and further tests will be required before definite conclusions on the
ability of animals to recover from Provera
and testosterone treatment are formed.
However, the results available from the
present study suggest that recovery of a
normal microscopic appearance of the testis is attained between three to six weeks
after the end of treatment. Taking into
account the length of the cycle of the
seminiferous epithelium in the rat (Clermont et al., '59; Steinberger and Steinberger, '75), about 12 days, or nearly two weeks,
would be required for spermatids to mature
once they were able to proceed normally
past spermatid stages 6-7. Since the testis
did not return to normal in three weeks,
but required between three and six weeks
to do so, there is apparently a lag period of
several weeks after the end of treatment
before spermatids begin to proceed normally past the cap phase. Apparently the
testis recovers first, followed by reversal of
epididymal alterations, and a return to
normal of the entire reproductive tract in
6 to 12 weeks.
The results suggest that infertility in
animals treated with Provera and testosterone is due in large part to an alteration
in spermatogenesis. Specifically this involves changes in the development of spermatids, beginning with the cap phase and
resulting in the degeneration of many
spermatids at this and subsequent stages.
However, as described in the following
paper (Flickinger, '77), alterations in the
epididymis may contribute also to infertility because of microscopic evidence of
masses of debris in the distal cauda epididymidis and indications of uptake of
luminal material by the epithelium of the
proximal cauda epididymidis.
ACKNOWLEDGMENTS
The author is indebted to Mrs. Jeannette
PROGESTIN AND ANDROGEN. I
Charlton and Miss Sharon Odum for technical assistance. This research was supported by a grant from the Population
Council (M74.82), a contract with the National Institute of Child Health and Human Development (NOl-HD-1-2506), and
a grant from N.I.H. (1 RO1 HD10073-01).
415
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PLATES
PLATE 1
E X P L A N A T I O N OF F I G U R E S
Figs. 1-3
Light micrographs of the testis of normal and treated rats.
Normal testis. Germ cells in different stages of development are present
including primary spermatocytes (P), early spermatids with round
nuclei (Q), and late spermatids with elongated and condensed nuclei
(S). X 300.
Testis of a rat treated with Provera for 16 weeks (Experiment 2 ) . Early
stages of germ cells are present, including spermatogonia ( G ) , primary
spermatocytes (P), and early spermatids ( Q ) ,but there are no late spermatids with condensed or elongated nuclei. The arrows indicate dense,
degenerating cells (fig.8). X 300.
Testis of a rat treated with Provera and testosterone for eight weeks
(Experiment 5). As i n the case of animals administered Provera alone,
the early stages of germ cells are present, including primary spermatocytes (P) and early spermatids (Q), but late spermatids i n the acrusome
and maturation phases are virtually absent. Lipid droplets (L) are found
near the basal lamina of the seminiferous tubules. A dense degenerating cell is indicated by the arrow. The few profiles of tails i n the lumen
may belong to early spermatids or to the small number of germ cells
that reach later stages i n treated animals. x 300.
418
PROGESTIN A N D ANDROGEN. I
Charles J. Rickinger
PLATE 1
419
PLATE 2
E X P L A N A T I O N OF FIGURE
4
420
Low power electron micrograph of the seminiferous epithelium of a
rat treated with Provera and testosterone for eight weeks (Experiment
5 ) . Sertoli cell cytoplasm (C), nucleus (N), and some of the characteristic junctional specializations (J) between Sertoli cells are visible, a s
well as a large primary spermatocyte (P).The organelles of the Sertoli
cell and the spermatocyte have a normal appearance. The presence of
a synaptinemal complex (arrow) in the nucleus of the spermatocyte
suggests that prophase of the first meiotic division proceeded in this
specimen. x 11,500.
PROGESTIN AND ANDROGEN. I
PLATE 2
Charles J. Flickinger
42 1
PLATE 3
EXPLANATION O F FIGURES
422
5
Basal portion of a seminiferous tubule from a rat treated with Provera
and testosterone for eight weeks (Experiment 5 ) . A normal-appearing
spermatogonium ( G ) is identified as a small, oval cell with a round
nucleus. It lies next to the basal lamina (B) of the tubule. Also present
i n this field are parts of a Sertoli cell (C) a n d the cytoplasm of a spermatocyte (P). X 10.000,
6
A portion of a late Golgi phase spermatid (Stage 3 ) from a rat treated
with Provera for four weeks (Experiment 2 ) . This cell was not altered
b y the treatment. It displays a round nucleus (N), large Golgi apparatus (G), and the acrosomal vacuole which contains a dense acrosomal
granule (A). x 18,000.
PROGESTIN A N D ANDROGEN. I
Charles J. Flickinger
PLATE 3
423
PLATE 4
EXPLANATION OF FIGURE
7
424
A c a p phase spermatid from a n animal treated with Provera a n d testosterone for 16 weeks (Experiment 3). In treated animals, some cap
phase spermatids such as this retained a normal appearance, but others
were necrotic (fig. 8 ) . Those with a normal structure, as i n this example, exhibited a round nucleus (N), prominent Golgi apparatus ( G ) ,
the characteristic acrosomal cap (A), and a rim of mitochondria around
the margin of the cell (M). Frequently a chromatoid body (B) was also
visible. X 12,000.
PROGES'TIN AND ANDROGEN. I
Charles J. Flickinger
PLATE 4
425
PLATE 5
EXPLANATION O F FIGURE
8
426
Electron micrograph of a degenerating or necrotic cell in the seminiferous epithelium of a rat treated with Provera and testosterone for eight
weeks (Fxperiment 4). The cell is abnormally dense and its organelles
are distorted. It can be identified a s a cap phase spermatid, possibly
stage 6 or 7. by the shape of the remaining acrosomal vacuole (A). The
degenerating cell is surrounded by Sertoli cell cytoplasm (C). At the
bottom of the field is a membrane-bounded structure that morphologically resembles a lysosome (D) and also lies within the cytoplasm of
a Sertoli cell. N. nucleus. X 13,500.
PROGESTIN A N D ANDROGEN. I
Charles J. Flickinger
PLATE 5
42 7
PLATE 6
EXPLANATION
428
O F FIGURES
9
Portion of a seminiferous tubule of a rat treated with Provera and testosterone for four weeks (Experiment 4). Parts of a late spermatid are
present within a mass of cytoplasm, but they lack their usual relation
to one another. Organelles present include the condensed nucleus (N),
coarse tail fibers (T),and a structure that is not identified with certainty but may be a misshapen acrosome (A). X 16,500.
10
Part of a Sertoli cell of a rat administered Provera for four weeks (Experiment 2 ) . Many large lipid droplets (L) are present in treated animals, and in some instances, as here, they are so large as to indent the
nucleus (N) and displace other organelles. A structure resembling a
lysosome is also present (D). The length of the bar is 1 fim. X 9,700.
PROGESTIN AND ANDROGEN. I
Charles J. Flickinger
PLATE G
429
PROGESTIN AND ANDROGEN. I
PLATE 7
Charles J . Flickinger
EXPLANATION OF FIGURE
11
430
Light micrograph of the testis of a rat permitted to recover for nine
weeks after the end of treatment for eight weeks with Provera and testosterone (Experiment 5). Numerous condensed nuclei and tails of late
spermatids ( S ) are present in the seminiferous epithelium. X 295.
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progestin, general, testis, malen, rat, influence, effect, structure, androgen, observations, trace, fine, reproduction
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