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Folliculogenesis in the ovary of the mature mouseA radioautographic study.

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Folliculogenesis in the Ovary of the Mature Mouse :
A Radioautographic Study '
TERRELL R. HOAGE AND IVAN L. CAMERON
Department of A n a t o m y , T h e University of T e x a s Health Science Center
at Sun Antonio, 7703 Floyd Curl Drive, S u n Antonio, Texas 78284
ABSTRACT
Studies on the growth sequence of follicles i n mature mice four
to six months old were conducted by giving three injections of 16 wCi tritiated
thymidine (3H-T)/gm body weight at four hour intervals over a period of eight
hours. Subsequent radioautographic analysis on ovaries obtained 1, 3 , 5 , 9 and
17 days after the last injection showed an overall follicle growth time of 17 to
19 days. The duration of the different follicle stage times were also estimated
from radioautographic data. Intense "-T incorporation was noted i n the developing antra and zona pellucida of follicles of animals sacrificed one and three
days after treatment suggesting that these areas serve as precursor storage sites
during development. Oocytes of follicles i n early antrum formation also showed
juxtanucleolar 3H-T incorporation concomitantly with rapid and massive oocyte
and follicular growth. The findings further indicate that the earliest follicular
cells surrounding the oocyte originate at some distance from the developing
primordial follicle.
Through light microscopy studies i t has
been established that primordial germ cells
in mammals are differentiated in early embryonic life (Hertig et al., '56), 'and that
after migration to the genital ridge, these
germ cells are the source of all the oocytes
in the adult ovary. During the twelfth day
of gestation in the mouse, clusters of
oogonia become incompletely surrounded
by follicle cells and in subsequent days
the number of follicle cells increases by
mitosis. This results in the oogonial clusters being completely surrounded and follicular vascularization begins (Odor and
Blandau, '69). Primordial follicles form a s
individual oocytes are surrounded by squamous follicle cells (Odor and Blandau,
'69) and develop so that in the prepubertal
mouse multilaminar follicles consisting of
as many as six to nine cellular layers develop in concert with the oocyte. This prepubertal follicular development does not
appear to be under anterior pituitary control (Greep, '63) which later plays a
regulatory role in synthesis, growth and
degeneration of the oocyte.
With the onset of estrus in the ovary of
the mature mouse, marked changes occur
in the number and sizes of follicles, as
well as in the growth rate of the follicles
ANAT. REC., 184: 699-710.
(Mandl and Zuckerman, '52; Pederson,
'70a). Growth rates of granulosa cells
along with the growth of whole follicles
have been extensively examined and categorized (Pederson and Peters, '68; Peters,
'69; Pederson, '70a,b). The above studies
utilized mice ranging in age from birth up
to three months of age. The mice were
killed from 1 to 48 hours after the last injection of tritiated thymidine (3H-T). The
above data provided an excellent baseline for the present radioautographic time
series studies on the proliferation of follicle cells using four to six month old mice
injected with 48 ,Ci "-T/gm body weight.
Peters ('69) and Pederson ('70a) have projected a follicle growth cycle duration of
17 to 19 days which is corroborated in the
time series data of the present study. In
addition to determining the granulosa cell
labeling characteristics, we have also determined the duration of each follicular
stage throughout the 17-day time period.
MATERIALS AND METHODS
Adult, female virgin mice of the A/Jax
strain, four to six months old and weighReceived Aug. 18, '75. Accepted Oct. 29, '75.
1 Supported by USPHS grants NS08949-06 and CA16831-01,
699
700
TERRELL R. HOAGE AND IVAN L. CAMERON
Two slides from each animal were seing between 22 and 28 gms, each received three subcutaneous injections of 16 lected at random and used for radioautopci /gm body weight of tritiated thymidine graphic analysis.
(3H-T) (specific activity 1.9 Ci/mM in
Differential counts o f follicles
0.9% saline, Schwartz-Mann) at four hour
Follicles were counted in each section
intervals. At 1, 3 , 5, 9 and 17 days after the
initial injection three animals were anes- and scored for the developmental stage as
thetized with amobarbital sodium and per- described by Peters ('69). Six stages were
fused through the left ventricle with a enumerated in classes as follows: ( 1 )
cacodylate buffered mixture of formalde- Stage 2 , oocyte surrounded by a single
hyde and glutaraldehyde modified from the layer of squamous follicle cells (fig. 1);
procedure described by Brightman and ( 2 ) Stage 3 , oocyte surrounded by a single
Reese ('69). A dilute solution of 1% form- layer of cuboidal cells (fig. 2 ) ; ( 3 ) Stage
aldehyde and 1.25% glutaraldehyde was 4, oocyte surrounded by two to three cell
administered at room temperature for two layers without antrum formation (fig. 3 ) ;
minutes without prior rinse. A concen- ( 4 ) Stage 5-6, two or more layers of cells
trated solution containing 4% formal- around oocytes with the onset of antrum
dehyde and 5% glutaraldehyde was then formation (fig. 4 ) ; ( 5 ) Stage 7-8, oocyte
perfused for 15 minutes following which with cumulus oophorus and corona radiata
the animal was undisturbed for one to surrounded by a distinct antrum (fig. 5 ) ;
three hours. Perfused whole animals were ( 6 ) atretic follicles were also counted and
stored in buffered formalin until target tis- were considered degenerating when pyknosues were excised. Ovaries were removed tic nuclei formed a distinct layer on the
and embedded in paraffin for light niicro- inner boundary of granulosa cells (fig. 6).
scopy. Five micron sections were cut and The presence of these pyknotic cells in
prepared as slide sets to include animals any stage caused it to be placed in the
killed 1, 3 , 5, 9, and 17 days after treat- atretic class. Stage 2 follicles exhibited no
ment as follows:
label and were not counted in total oocyte
1. Sections of the ovary were placed on determinations.
slides subbed in 0.05% agar, subsequently
Silver grain count methods
deparaffinized, and air dried.
2. One set of slides was subjected to
Background counts were determined by
treatment in 5 N HC1 for one hour, then counting ten of one hundred 0.03 mm
exposed to Schiff's reagent for one hour. squares circumscribed by a calibrated
These slides were evaluated for DNA stain- ocular graticule. Background counts were
ing after the above Feulgen reaction.
taken over open slide areas at the border
3. Four additional sets of the slide series of tissues. These counts were then extrawere dipped in Kodak NTB-3 liquid radio- polated to a standard area that could be
autographic emulsion along with the Feul- used to compare the cytoplasm and nuclei
gen stained slides mentioned above.
observed in the study. All further deter4. One set of slides was digested in minations were made on counts above this
DNase (Worthington Biochemical, 10 mg background level. Antrum counts were
in 35 ml Tris buffer, pH 7.2) for eight made as described above and recorded in
hours while a comparison set remained in terms of grains per unit area as in backTris buffer.
ground determinations. Diameters were
5. All dipped slide sets were allowed to determined on follicles cut in sagittal secair dry and were placed in light tight slide tion and with oocytes showing nucleoli
boxes and stored at 4 ° C for three weeks within the nucleoplasm. This insured a
of exposure.
relative maximum for both cell and nu6. After three weeks of exposure the clear diameter. The outside diameter of the
slides were developed in Dektol and stained zona pellucida ( Z P ) was taken and used
with hematoxylin and eosin, except for the along with the oocyte diameter to deterFeulgen stained slides which were counter- mine the area of the ZP on which total
stained with Fast green.
grain counts were taken.
MOUSE OVARIAN FOLLICLES
Fig. 1 Stage 2 primordial oocyte ( 0 ) of mouse surrounded by squamous follicle cells ( F ) ,
stroma cells ( S ) . x 1,400.
Fig. 2 Mouse primary oocyte of stage 3 with a layer of cuboidal follicle cells ( F ) surrounding the oocyte ( 0 ) . x 1,200.
Fig. 3 Follicle stage in transition from 3 to 4 with a differentiating thecal (T)layer at
the periphery of the mouse follicle cells ( F ) , oocyte ( 0 ) . x 1,200.
70 1
702
TERRELL R. HOAGE AND IVAN L. CAMERON
703
MOUSE OVARIAN FOLLICLES
Numbers of Follicles in Mouse Ovary Sections
Expressed in Percent for Each Stage Scored
r
~
+ 100I
0
a
-
W
80v)
W
- 1
0
-
60-
e -
T
_I
40F
L
0
5
L
-
20-
w
0
-
LT
W
L
O
/
0
&
I
I
2
I
1
4
I
l
l
6
I
8
I
I
10
1
I
12
l
l
14
I
I
16
1
DAYS AFTER 3H-THYMIDINE INJECTION
Fig. 7 Average frequencies of follicular stages of mouse ovaries at 1, 3, 5, 9 and 17 days
after injection of 3H-T.
Photomicrographs were taken on a Zeiss
microscope using either Panatomic-x or
high contrast copy film which was subsequently developed in Microdol-x or D-19
respectively.
RESULTS
Differential follicle counts
The differential follicle count present
without reference to labeled cells, in the
stages described above, are recorded in
table 1. Follicles in stage 3 were most numerous in sections obtained one and nine
days after sacrifice. Stages 4 and 5-6 were
not significantly different from each other
while the stage 7-8 follicles were significantly less than all of the other classes in
all sections counted. The average change
in follicular types with time after 3H-T in~
~~
Fig. 4 Stage 5-6 mouse follicle demonstrating
concentrated "-T label i n the antrum ( A ) , zona
pellucida ( Z ) and nuclear ( N ) label of actively
synthesizing follicle cells and the oocyte (0).
x 800.
Fig. 5 Follicle i n stage 7-8 with fully developed oocyte ( O ) , Nucleus ( N ) , antrum ( A ) .
x 250.
Fig, 6 Atretic stage 7-8 follicle as noted by the
layer of pyknotic nuclei (arrows) bordering the
antrum ( A ) , oocyte (0). x 250.
jection are shown in figure 7 indicating a
decline in the proportional number of functional follicles over the 17 day test period,
in the animals studied. I n conjunction
with this decline there is a significant increase in atretic follicles over time (table
1).
Follicle labeling patterns
No labeled cell nuclei were observed in
stage 2 follicles at any time. Of the remaining follicle stages, it was estimated that
90% of the observed follicles possessed
one or more heavily labeled theca and
granulosa cells from one to five days after
"-T administration. The observed label
varied from an estimated 150' grains per
nucleus in cells of animals sacrificed one
and three days after injection to the five
grains or less observed per nucleus in animals sacrificed 17 days after JH-T injection. This reduction in grains per nucleus
is expected i n rapidly proliferating cell
populations such as found in oocyte follicular development. Beginning with 150'
grains/nucleus it would require six doublings to reduce the grain count to less
than 5 grains/nucleus. This dilution effect
coupled with peak labeling percentages
observed in granulosa cells (table 2, fig. 8 )
704
TERRELL R. HOAGE AND IVAN L. CAMERON
TABLE 1
Differential follicle counts o f four t o six m o n t h old m o u s e ovaries t a k e n at 1, 3, 5, 9 a n d
17 days a f t e r injection o f 48 Fci " - T / g m body weight over a n eight hour period
Stage of follicle development
Day(s) after
YH-T injection
1
3
5
9
17
1 Mean
Stage 3
Stage 4
Stage 5-6
Stage 7-8
Atretic
36.221.21
27.820.61
20.420.95
30.420.98
12.720.94
18.2t1.04
25.220.89
22.421.32
19.2'0.81
14.011.51
23.2k0.82
29.0'1.29
24.5k1.55
17.620.77
23.0e1.31
12.020.64
1O.OkO.59
8.8k0.91
7.620.67
3.820.85
10.520.71
8.3C1.25
37.222.73
25.2-tO.97
47.3%1.71
-I- S.E.M.
TABLE 2
Percent o f granulosa cells labeled per follicle in ovaries o f f o u r to six m o n t h old mice
injected with 48 yci 3 H - T / g m body weight over a n eight hour period a n d sacrificed
I , 3, 5 , 9 a n d 17 days a f t e r treatment
Stage of follicle development
Day(s) after
3H-T injection
0%
1
3
5
9
17
1
Mean
Stage 4
Stage 5-6
Stage 7-8
4 2 . 6 1 1.47
7 1 . 3 t 1.24
89.5 -C 1.81
94.3 2 0.62
35.72 1.35
41.22 1.55
59.2 t 2.03
7 8 . 4 r 1.34
100.0 2 0.00
21.620.75
51.1 &2.36
98.0 2 0.84
68.0-t 1.71
64.0 2 1.88
00.0t0.00
Stage 3
59.5? 3.14
80.0 2 1.56
70.72 0.92
2.4 t 0.82
S.E.M.
a
cn
J
B
gE
UJ
V W
rrm
WQ
CLJ
0
2
4
6
8
10
12
14
16
DAYS AFTER 3H-THYMIDINE INJECTION
Fig. 8 Mouse ovary follicle granulosal label b y stage over a t i m e series o f 1, 3, 5, 9 and
17 days after SH-T injection.
were used to estimate time periods for the
development of the various follicle stages.
For example, stage 3 follicles exhibited the
highest frequency of labeled cells on the
fifth day after 'H-T injection (table 2, fig.
8 ) and the number of grains per nucleus
were approximately one half that observed
at day 3 . This indicates division of cells
either within the follicle or within a labeled
cell population serving as precursors to the
granulosa cells.
The label over granulosa cells of all
705
MOUSE OVARIAN FOLLICLES
follicular stages in animals sacrificed at
9 and 17 days was further reduced in
grains/nucleus and the number of cells
counted as labeled declined even more.
Such a decline in the percentage of labeled cells would be expected by halving of
grains nucleus over six or more cell cycles.
Stages 4 and 5-6 showed a peak in percent
of labeled cells on the ninth day (table 2 )
after isotope injection, which was interpreted as the result of proliferation from
highly labeled cells present immediately
after ,'H-T injection. This proliferation of
labeled cells is confirmed by the reduced
number ol grains per nucleus over cells at
day nine. Further proliferative activity
causes stages 4 and 5-6 follicles to grow
into the mature stage 7-8 follicles. Stage
7-8 follicles counted on days 5, 9 and 17
decline in number of grains per nucleus
and concomitantly in the number of nuclei
designated as labeled (table 2, fig. 8 ) .
Following the above observations on
grains per nucleus and percent of labeled
cells in each stage scored, it is concluded
that stage 3 follicles stimulated to growth
required from day 5 to 17 to complete
growth of labeled follicles into stage 4 or
later. Stage 4 follicles maintain a high percentage of labeled nuclei on days 3, 5 and
9 (fig. 8 ) and then decline to less than day
1 levels by day 17. Such a sequence indicates that stage 4 follicle cells labeled at
day 1 are advanced to later stage 4's until
eventually all stage 4's have moved on to
stage 5-6. This dilution in percent of labeled cells by division which occurs after
day 9 indicates a 7-day maximum duration
for stage 4 to grow into stage 5-6. The
increase in percent of labeled cells which
occurs between three to past nine days of
stage 4 follicles indicates a 6-day time duration for stage 4 follicles to grow into stage
5-6 follicles. Stage 5-6 follicles are somewhat more difficult to interpret due to the
greatly reduced number of grains over
nuclei on days 5 and 9. This rapid dilution
of grains nucleus in labeled cells indicates
that the cell populations of stage 5-6 seen
on day 3 are different follicles than the
stage 5-6 follicles seen on days 5, 9 and 17.
Therefore those stage 5-6 follicles seen on
days 5, 9 and 17 must have originated
from a n earlier follicle stage which possessed a high grain count per nucleus on
day 3 . Thus i t can be concluded that stage
5-6 follicles move into stage 7-8 between
days 3 and 5 thus suggesting a duration of
two days or less. The above is also true of
mature stage 7-8 follicles that go to almost
100% labeled cells from day 1 to day 3.
The loss of stage 7-8 follicles to ovulation
results in immediate replacement from
stage 5-6 with a concomitant loss in number of grains nucleus in these new stage
7-8 follicles until by day 17 the number of
grains/nucleus in labeled cells is less than
or equal to background label. This indicates a maximum time of less than two
days for stage 5-6 follicles to move into
stage 7-8 and then out. It also indicates
that at least six cell cycles have occurred
during the growth of early follicle stages to
the mature stage.
Thecal cell development
In early follicular development the type
of cells associated with the oocyte is limited to the squamous cell type which in the
later follicular stages become either theca
externa and theca interna or granulosa
cells. The degree of labeling per follicle, ex-
TABLE 3
Percent of thecal cells labeled per follicle i n sections of ovaries of f o u r to six m o n t h
old m i c e injected w i t h 48 pci s H - T / g m body weight and sacrificed 1 , 3 , 5, 9
and 1 7 days a f t e r treatment
Stage of follicle development
D a y ( s ) after
3H-T injection
1
3
5
9
17
1
Mean & S.E.M.
Stage 3
00.0 & 0.00
38.3 2 2.31
36.0t 2.44
54.7t2.42
00.0"0.00
Stage 4
37.71 1.30
42.8% 1.28
46.8% 2.70
85.0% 1.30
16.3% 1.57
Stage 5-6
38.811.50
40.8-C 1.48
58.6 -C 1 .80
64.7k 3.22
8.3 k 1.07
Stage 7-8
32.6t 1.70
51.822.31
59.72 2.30
88.0% 1.33
4.0 i 1.52
706
TERRELL R. HOAGE A N D IVAN L. CAMERON
Stage 78lnterna
W
0
Stage 4
a
J
Stage 7-8 Externa
0
5-6
[L
I
5
3
9
17
DAYS AFTER 3H-THYMIDINE INJECTION
Fig. 9 Percent labeled thecal cells in follicular development of four to six month old mice
treated with 3H-T and sacrificed 1, 3, 5, 9 and 17 days after injection.
TABLE 4
Follicle antrum label in sections of ovaries of four to six month old mice injected with
48 pci SH-T/gm body weight and sacrificed 1, 3, 5, 9 and 17 days after treatment
Stage of follicle development
Day(s) after
3H-T injection
1
3
5
9
17
1 Mean
Stage 5-6
Stage 7-8
115% 1.73
39 i 1.48
29 f 1.55
3 1 1.11
27 f 0.86
146 C 2.32
58C2.18
27 t 0.78
2 9 r 1.33
23 t 0.58
*
Background label
25 % 0.96
23 % 1.49
28 C 0.74
2 2 2 1.85
24 i0.89
f S.E.M.
pressed in average percent of thecal cells
labeled is given in table 3. Cellular proliferative activity expressed by the labeling
index was not seen in stage 3 of day 1.
Stage 3 thecal cells reached a labeled maximum on day 9. It appeared that those cells
labeled in the various stages other than
stage 3 increased slowly in label to a maximum on day 9 with rather severe drops
in label by day 17 (table 3 , fig. 9 ) . This
can be accounted for by the development
of follicles stimulated to maturity within
this 17-day test period. The subsequent
ovulations (four estrous cycles within this
test period) would account for the thecal
label decline as would dilution through
several division of cells labeled very early
in the follicle cycle activities. Theca externa cells of stages 5-6 and 7-8 exhibit
rather rapid dilution of label which is
expected in rapidly proliferative cell populations.
Of particular interest is the absence of
label in the early stage 3 follicle cells one
day after "-T injection and the subsequent
appearance of labeled thecal cells in later
stage 3 development which occurs several
days after the 'H-T injection. Such a labeling pattern of early stage 3 follicle cells
suggests migratory activity of labeled cells
to the oocytes by either labeled stroma cells
or by some other labeled cell from a population extrinsic to the ovary.
Antrum development
Antrum development is initiated in late
stage 5 follicles. In those antral follicles
active at the time of "-T injection very intense label is found at day 1 (table 4 ) .
Both stages 5-6 and 7-8 exhibit the concen-
MOUSE OVARIAN FOLLICLES
i -\
I
3
5
9
I7
DAYS A F T E R 3H-THYMIDINE INJECTION
Fig. 10 Graph showing radioautographic label
lost per unit area of antrum in follicles of four to
six month old mice 1, 3, 5, 9 and 17 days after
3H-T injection.
tration of radioactive material at highly
significant levels above background label.
Similar counts are also observed in the ZP
of these follicle types. The fate of this precursor is not obvious within this study but
the 3-day antral follicles maintained a level
of label that was still significantly above
background although not as high as day 1
antra. Days 5, 9 and 17 did not show label
above that observed for background (fig.
10). Enzymatic digestion with DNase did
not significantly decrease the antrum label
from 115 and 146 grains in stages 5-6 and
7-8 respectively. Since this level was not
affected by DNase digestion, the 'H-T or its
metabolites are bound in material other
than DNA.
DISCUSSION
The frequency of specific follicle stages
has been delineated by Pederson ('70a,b)
and the follicles were generally categorized
into small follicles (stages 1, 2 , 3 a ) , medium follicles (stages 3b, 4), large follicles
(stages 5, 6 ) and preovulatory follicles
(stages 7, 8). These categories correspond
to the relative stages described in this
study. It is deduced from this study that
type 2 follicular cells have their origin in
the vicinity of the type 2 follicles observed.
The statement that type 2 follicles do not
grow but are formed (Pederson, '70a) is
apparent in previous studies from the lack
707
of incorporation of 'H-T into the squamous
cells seen in primordial follicles (Pederson,
'69). Peters and Pederson ('67) state that
the origin of these cells would appear to be
from the ovarian stroma although the site
of label and interim location were not identified, Labeled stroma cells were observed
by the previous authors but at great distances from the oocyte nests. Since the
origin of both thecal cells and follicle cells
cannot be ascertained with certainty, further studies are needed to identify the
sources and to determine both the stimulus
and migratory time to initiate a shift from
the nongrowing stage 2 follicle type to
the stage 3 which is a growing follicle
type. Parabiosis with 'H-T radioautographic
studies is in progress to clarify this phenomenon.
Our follicular numbers, types, duration
of developmental stages as well as the total
follicle development time are within the
same range as depicted by Pederson ('70a).
There is, however, a distinct increase in
atretic follicle number over the time span
of this study and several things could account for this increase atresia. It has been
shown that high levels of incorporation of
'H-T ( 1500 @/pregnant mouse over a 120
hour period) cause oocyte degeneration if
incorporated during embryonic growth
(Callebaut, '68). At four months of age
these female mice had ovaries with no
functional oocytes and many follicles that
did not complete development. This then,
suggests a degeneration of follicles if the
oocyte is damaged. Such radiation damage
to cellular function could account for the
increased atresia of follicles noted in the
latter days of this experiment. Also the
fact that oocytes do incorporate 3H-T into
juxtanucleolar DNA (Hoage and Cameron, unpublished observations; Crone and
Peters, '68) could also account for the increased atresia on the basis of radioactive
damage. It is also possible that the incorporation of 3H-T into secretory cells of the
pituitary could affect hormone levels that
lead to atresia in the small growing oocytes
and follicles (Jones and Krohn, '61; Pavic,
'63). Pederson ('69) concluded that only
large follicles degenerate which was not
the case in this study where pyknotic nuclei
occurred in medium sized follicles comparable to stage 4. This variation can also be
708
TERRELL R. HOAGE AND IVAN L. CAMERON
attributed to the effects of increased 'H-T
incorporation in the nucleus of stage 5
oocytes (Hoage and Cameron, unpublished
observations.
It has been shown that baseline estimates of pulse labeled follicles established
by Peters ('69) and Pederson ('70a) are
accurate when determined over longer
sampling times ( u p to 17 days after 'H-T
application). The total time determined in
this study for follicular growth from stimulated stage 3 through to ovulation was
17-19 days which agrees with previous
studies (Pederson, '70a,b).
Thecal cell development has been followed in this study and the findings indicate that the precursors to thecal cells originate from a population of labeled cells
exterior to the primordial follicle. The
source of the thecal cells is not apparent
from this examination but the source appears to be drawn from either the ovarian
stroma or another undifferentiated cell
population with migratory capabilities. The
ultrastructural similarity of lymphocytes
and secretory thecal cells and granulosa
cells (Weakly, '66) suggests that lymphocytes may be a source.
Antrum development through fluid production has been well documented (Hertig
and Barton, '72, for review) but the role of
the antrum is not clear with respect to development of follicle cells and oocyte activities. The presence of extremely high levels
of radioactive label in antra shortly after
injection of 3H-T suggests a reservoir of
precursors for the rapid proliferative activity that accompany antra and follicle
growth in these stages. Concurrent with
the growth phase there is an increase
in oocyte nuclear synthetic activity that
involves juxtanucleolar DNA synthesis
(Hoage and Cameron, unpublished observations) that could draw on nucleoside reserves present in the antrum. That this
antrum labeling is not DNase sensitive
indicates that the material is not incorporated into DNA. Routine histological
processing might be expected to extract
soluble JH-T,however, it is conceivable that
soluble ,'H-T was in some way fixed to
higher molecular weight material at the
time of glutaraldehyde fixation in a manner similar to the fixation of an amino acid
to macromolecular material as shown by
Peters and Ashley ('67). The state of the
tritium label in the antrum is not clear but
it must be highly compact and or complexed to ellicit the concentrated response
seen in this study. The increased thecal
development concurrent with the antrum
label suggests a coordinated transport system to augment the rapid growth sequence
of the maturing follicles.
ACKNOWLEDGMENTS
The authors wish to thank Dr. E. I<.
Adrian for his cooperation and for generously supplying the animals used in this
study. We also appreciate helpful discussions about the manuscript with Dr. E. G.
Rennels and the excellent assistance of W.
A. Pavlat.
LITERATURE CITED
Brightman, M. W., and T. S. Reese 1969 Junction between intimately apposed cell membranes in the vertebrate brain. J. Cell Biol., 40:
648-677.
Callebaut, M. 1968 Development of the gonads
of mice after intense incorporation of tritiated
thymidine during the period of oogenesis. Experientia, 24: 828-829.
Crone, M., and H. Peters 1968 Unusual incorporation of tritiated thymidine into early diplotene oocytes of mice. Exp. Cell Res., 50: 664668.
Greep, R. 0. 1963 Histology, histochemistry
and ultrastructure of adult ovary. In: The
Ovary. H. G. Grady and D. E. Smith, eds. Williams and Wilkins, Baltimore, pp. 48-68.
Hertig, A. T., and B. R. Barton 1972 Fine structure of mammalian oocytes and ova. In: Handbook of Physiology-Endocrinology 11-Female
Reproductive System Part I .Vol. 7. American
Physiological Society, Washington, D.C., pp.
3 17-348.
Hertig, A. T., J. Rock and E. C. Adams 1956 A
description of 34 h u m a n ova within the first 17
days of development. Am. J. Anat., 98: 435-493.
Jones, E. C., and P. L. Krohn 1961 The effect
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