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Follicle Growth and Cell Dynamics in the Mouse Ovary During Pregnancy
Vol. 22, No.1, January 1971
FERTn.rrv AND STERn.1TY
Copyright
©
Printed in U.S.A.
1971 by 'The Williams & Wilkins Co.
FOLLICLE GROWTH AND CELL DYNAMICS IN THE MOUSE OVARY DURING PREGNANCY 1'-
TORBEN PEDERSEN
AND
HANNAH PETERS
The Finsen Laboratory, The Finsen Institute, Copenhagen, Denmark
It has recently become possible to fol- ments. They were mated, and the onset low the development and movements of of pregnancy was determined by the prescell populations in the ovary for a consid- ence of a vaginal plug (Day 0 of pregerable period of time in autoradiographs nancy). The average length of pregnancy after pulse labeling with tritiated thymi- is 19-20 days in this strain of mice. All mice were pulse-labeled by injectdine. 1 Moreover, the number of follicles that begin to grow at any time unit and ing intraperitoneally 100 JLC. of tritiated the speed of their development can be thymidine (H 3 TdR; Amersham Radiodetermined with follicle kinetic studies. 2 chemical Center, specific activity ranging The purpose of this investigation was to from 3.6-5.0 c./mM) either before or follow follicle growth and development during pregnancy. The animals were in the mouse ovary during pregnancy. killed at different time intervals after the Dempsey3 stated that pregnancy does not injection. Autoradiographs (ARG) were interfere with follicle growth in the guinea prepared of the serially sectioned (5 JL) . pig. Greenwald and Choudary4 reported a ovaries with K2 emulsion (Ilford), and extemporary decline in follicular activity in posed for 1-4 weeks at 4° C. They were the mouse ovary at the end of the second developed and then stained with hemaweek and a resumption of development at toxylin-eosin. At least 2 mice were used the beginning of the third week of gesta- for each determination. In the ARG of the ovaries, the labeling tion. In order to elucidate the complexity of follicle growth during pregnancy, the pattern as well as the labeling index of number of follicles which begin to develop the different structures was determined. during the different stages of pregnancy The distribution of labeled cells within a as well as their speed of growth was inves- structure (the follicle or the stroma reptigated. Furthermore, it seemed of interest resents the labeling pattern of the structo know whether those follicles which be- ture. The labeling index (LI) of a follicle gin to grow grow continuously or whether was defined as the number of labeled their development is held in abeyance at granulosa cells in percentage of the total any time. Finally, it seemed relevant to ob- number of cells; it was determined in the serve the fate of those developing follicles ARG 1 hr. after pulse labeling in that and corpora lutea which are already pres- cross-section of a follicle in which the ent in the ovary at the time that preg- nucleolus of the oocyte is seen. 2 nancy is initiated. These questions were To describe follicle growth a classificainvestigated in consecutive autoradio- tion of follicles based on the morphology graphs of ovaries of pregnant mice after and the number of granulosa cells was pulse labeling with tritiated thymidine. used5 (Fig. 1). Follicles of Type 3b were counted in MATERIALS AND METHODS every fifth section of the serially sectioned Sixty nulliparous Bagg mice, 3-4 ovary using the nucleolus of the oocyte as months old, were used in these experi- a marker. 42
'f
..
..
January 1971
(4~
~------
,
..... -
,,~t? ,// ¢V~ __ / 0" ",f:l~,/ TYPE
~ Iv'" /'
TYPE 3b
~ S~~ -' 'OJ
:
\
TYPE 3a
\,
0
~ 0 ~
;.
lSI
'.
"-'"
-'''-.
......
401-600 CEllS
61-100 CEllS >600 CEllS
~
'GI
\\'\,
.
TYPE 7
\\
\\\,
\.
e ! 20 CEllS
r, \
..J
(~s
--_" "P.J
TYPE 6
a~\ u..
~(/c '>0
21-60 CEllS
!
If)
~
G
oj,
---_
201-400 CEllS
~ 101-200 CEllS
TYPE 4
--____~yl'~
o
0 0
;j' ...." ./ ~ OV , /
/
50
--------__________ (OQG~ ....0
TYPE 5b
__ ---_7/;/'
~""\;;"O,',//
43
FOLLICLE GROWTH DURING PREGNANCY
TYPE 8
TYPE 2 'IS\' 'i?
\ "
\. ...
~
~
TYPE 1 @
FIG. 1. Classification of follicles
In order to determine the evolution of follicles and the fate of corpora lutea during pregnancy several experiments were performed.
The Growth of Follicles during Pregnancy Injection of HJTdR on Different days of Pregnancy. Twenty-two mice were injected with H 3 TdR on 1 of the following days of pregnancy: 2, 4, 8, 10, 12, 17, or 20. The animals were killed 1 hr. later and an ARG was prepared of their ovaries (immediate ARG). The labeling patterns of follicles and stroma, as well as the labeling index of follicles in different stages of development, were determined. The labeling pattern of follicles gave an indication of their growth: follicles containing labeled granulosa cells were proliferating while those without labeled cells were not. A change in the LI of a given follicle type indicated a change in the growth rate. 2 If, at any day, "healthy" medium or large follicles which on previous days had incorporated the label failed to do so, it was suggestive that the growth of that
follicle type had been held in abeyance. The labeling pattern of the stroma was observed to determine whether the stroma proliferated during pregnancy. Injection of 2 Different Doses of lfTdR on Day 12 of Pregnancy. In order to test the speed of follicle growth, the duration of the deoxyribonucleic acid (DNA) synthesis (S-phase) in granulosa cells was estimated on the 12th day of gestation. This was done indirectly, using the double labeling technic. s - 9 Two markedly differing doses of H 3TdR were used to produce heavily and lightly labeled cells in the ARG. Two mice were injected on Day 12 of pregnancy with 10 p.C. of H 3TdR and 1 hr. later with 100 p.C. of H 3 TdR. The animals were killed 45 min. after the last injection. For comparison, two 28-day-old mice which were not pregnant were treated in the same manner. The actual duration of the S-phase in granulosa cells had been previously determined in 28-day-old mice, using the method of labeled mitosis1o ; the results were used for comparison in this study. ARG's prepared after double labeling were exposed for 14 days. The number
44
PEDERSEN AND PETERS
of faintly labeled cells (up to 15 gr. over the nucleus) in percentage of heavily labeled cells was determined in the different follicles. Injection of lfTdR on the Day of the Vaginal Plug (Fig. 2). To follow the formation of the peripheral stroma, the following experiment was done. Twenty mice were mated, and, on the day of3 the vaginal plug (Day 0 of pregnancy), H TdR was injected. Autoradiographs were prepared at intervals of 1 hr. and 1, 2, 3, 4, 6, 8, 12, 17, and 20 days after pulse labeling. In the immediate ARG, proliferating and nonproliferating cell populations were determined. In autoradiographs prepared some days after the injection of H 3TdR (delayed ARG), the evolution of those structures that had incorporated the label at the beginning of pregnancy could be followed. A displacement of labeled cell groups became evident and, furthermore, cell divisions could be inferred if heavily labeled cells became replaced by lightly labeled cells. VAGINAL PLUG 3H-TdR
~----CORPUS LUTEUM OF PREGNANCY
LARGE FOLLICLE
0)-----GROWING FOLLICLE
@--------SMALL FOLLICLE
FIG. 2. Schematic illustration of label distribution in the ovary after pulse labeling on the day of the vaginal plug.
Vol. 22
r~
The Fate of the Prepregnancy Corpora Lutea
Injection of H 3 TdR before Conception (Fig. 3). In order to distinguish between the corpora lutea of pregnancy and those formed in cycles before conception and to follow their persistance during pregnancy, 16 mice were injected on the day of estrus (Eo) and mated 2 days later. Mice that showed a vaginal plug within 2 days were sacrificed on Days 2, 4, 6, 8, 12, 14, 18, and 20 of pregnancy for the preparation of an ARG of their ovaries. In such an experimental set-up, the corpora lutea of pregnancy and those of prepregnancy cycles can be distinguished, as the corpus luteum of pregnancy contain labeled cells, while the corpora lutea that were already in the ovary at the time of injection (corpora lutea of Eo and EO-I) remain free of the label. I RESULTS
Follicle Growth.during Pregnancy The immediate ARG obtained after injection of H3'J'dR on different days of pregnancy clearly showed the proliferating and nonproliferating cell systems in the ovary (Fig. 4). Cells of the medium and large follicles are proliferating while the cells of the stroma and corpora lutea are not. The number of small, Type 3a follicles that showed labeled granulosa cells varied from 20% at the beginning (Day 2) to 40% at the end (Day 20) of pregnancy (Table 1). However, all the medium and large follicles showed labeled granulosa cells after pulse labeling on the different days of pregnancy, indicating that DNA synthesis took place on all days tested. This suggests that all healthy medium and large follicles grow, and that none stops growth temporarily to resume it later in pregnancy. Follicles in degeneration as well as contracted follicles l l were free of labeled cells. Stroma cells were unlabeled in all immediate ARG's. However, in
\r
January 1971
45
FOLLICLE GROWTH DURING PREGNANCY
PREGNANCY
MALE IN CAGE
OESTRUS (Eo)
!
o
CORPUS LUTEUM OF EO-1
CORPUS LUTEUM OF EO
-~~-.
OESTRUS (E,)
o
o
.~ . ..;;~, .. ---® .
"
. ".~'''.'
. LARGE FOLLICLE
-
.
CORPUS LUTEUM OF PREGNANCY
@ GROWING FOLLICLE
@ SMALL FOLLICLE
LARGE FOLLICLE
@
-
GROWING FOLLICLE
FIG. 3. Schematic illustration of label distribution after pulse labeling at estrus and label dilution several days later at the time of fertilization.
ARG's prepared 6 days or later after pulse servations suggest that part of the periphlabeling on Day 0, areas of labeled cells eral stroma seen during pregnancy derives were seen in the peripheral stroma. These from contracted and transforming follicles. In order to define follicle growth in areas were numerous during midpreggreater detail, two factors are discussed: nancy and became fewer toward the end. (1) the speed with which they grow, in The cells in such areas were usually particular, the changing rate of growth lightly labeled, suggesting that they must during their development and (2) the have belonged to a cell population which, that begin to grow per number of follicles since pulse labeling, had divided and diunit time during pregnancy. luted the label. Delayed ARG's prepared The speed of growth of a follicle de6 or more days after pulse labeling in midpends upon the proliferation rate of its pregnancy also showed areas of -labeled granulosa cell population. This is detercells in the peripheral stroma. As only the the number of cells synthesizing mined by granulosa cells of medium and large folliDNA (expressed as the LI of the follicle) cles are proliferating during pregnancy, it and by the duration of the S-phase of the is inferred that the areas of labeled cells single granulosa cells. The LI in the varin the stroma are derived from cell groups ious types of growing follicles was deteroriginally belonging to follicles. These ob-
46
PEDERSEN AND PETERS
Vol. 22
;.-.
,
FIG. 4. Labeling pattern of an ovary on the 12th day of pregnancy 1 hr. after injection of H'TdR. Granulosa cells incorporated the label while the cells of corpora lutea and stroma remained unlabeled. X 170.
TABLE 1. Percentage of Small Follicles (Type 3a) Containing Labeled Granulosa Cells on Different Days of Pregnancy and during the Cycle in ARG Prepared 1 hr. after Pulse labeling No. of small
H'TdR injected
follicles (Type 3a) with labeled granulosa cells
On day of pregnancy 2 4 8 10 12 17 20 (day of litter birth) During estrus cycle
%
21
32 28 36 34 34
40 34 (mean value)
mined at different times during pregnancy and compared with those found in the nonpregnant mouse during a cycle7 (Table 2). The LI of large follicles was considerably higher than in medium follicles throughout pregnancy as well as during the estrus cycle. Considering the different
follicle types during pregnancy, it is noted that the LI in the Type 3b follicle is unaltered throughout pregnancy, while the LI of Types 4, 5a, and the large follicles tends to decrease temporarily at midpregnancy; the differences in the values are, however, statistically not significant at a 5% level. The LI in comparable follicle types during pregnancy and during the cycle are similar, indicating that the number of granulosa cells synthesizing DNA in comparable follicles is similar during pregnancy and during the cycle. The duration of the S-phase was investigated indirectly for medium and large follicles on the 12th day of pregnancy. In ARG's prepared after 2 differing doses of H 3 TdR (double labeling experiment) the percentage of faintly labeled cells in relation to heavily labeled cells was determined for follicles in 3-month-old pregnant mice as well as in 28-day-old virgin mice. A comparison of the percent of lightly labeled cells in the two groups of mice permitted a comparison of the dura-
..
.....
Jarwary 1971
47
FOLLICLE GROWTH DURING PREGNANCY
~
TABLE 2. Labeling Index of Follicles on Different Days of PreglUlncy and during the Estrus Cycle
...
Large foDicles
Medium foDicles
""
Mean
~
%
":,
-. -< "-
Day of pregnancy 2 4 8 10 12 17 20 (day oflitter
Type 5a
Type 4
Type 3b
S.E.
Mean
Mean
S.E.
S.E.
Mean
S.E.
'1 ~
S.E.
Mean
S.E.
%
9.2 9.1 8.4 8.6 8.5 9.1 9.6
0.9 0.8 1.2 1.2 0.8 0.7 0.9
7.7 7.5 6.0 5.6 7.1 6.0 7.4
0.9 0.8 0.9 0.7 0.5 0.6 0.7
15.6 15.2 16.1 11.5 11.7 11.6 20.9
3.2 2.3 2.7 3.4 1.5 1.5 3.0
42.1 25.8 23.8 29.3 27.9 35.7 42.9
3.4 2.7 2.5 2.1 2.0 1.6 3.0
22.2 25.0 44.1* 27.7 24.2 43.6*
0.8 4.4
9.3
0.2
7.3
0.2
15.3
0.5
35.8
1.0
35.2
1.4
birth)
Mean LI during estrus cycle
Mean
Type 7
%
%
%
%
Type 6
Type 5b
4.0 2.8
22.3 20.9 15.9 20.0* 16.4 16.8 22.2
1.7 8.7 2.4
28.0
1.5
1.9 2.2 8.7
* Based on one detennination only.
TABLE 3. Percentage of Faintly Labeled Cells in Follicles after Injection of Two Different Doses (10 ,.c. and 100 ,.c.) of lfTdR on the 12th Day of PreglUlncy and in 28-Day-Old Virgin Mice Medium foDicles
Large foDicle.
Type 3b Type 4 Type 5a Type 5b Type 6 %
12th day of pregnancy \3month-old mice) 2S-Day_old-virgin mice
13.5
14.5
13.9
18.5
17.7
14.7
13.0
13.9
16.8
16.5
9.8
11.2
hr. Duration of S-phase in granulosa cells determined in 28-day-old
10.3
6.8
6.8
virgin mice7
tion of the S-phase, as the following relationship exists9 : t.
Nh Nf
=
ta
where Nh is the number of heavily labeled cells, Nt is the number of faintly labeled cells, t. is the duration of the Sphase, and ta is the interval between injections. The values for the percentage of faintly labeled cells in comparable follicle types
in the two groups of mice were obtained (Table 3). They were not different at the 5% level, suggesting that the DNA synthesis time is comparable and that no large variation in the duration of DNA synthesis time in comparable follicles has occurred on the 12th day of pregnancy. In order to determine the number of follicles that begin to grow during a 24-hr. period, two factors have to be known: (1) the number of Type 3b follicles present in the ovary and (2) the transit time,2 i.e., the time that it takes a 3b follicle to grow from one with 21 to one with 60 granulosa cells on its largest cross section (Fig. 1). 1. The total number of Type 3b follicles present in the ovary of mice at different stages of pregnancy was determined and compared with the number of Type 3b follicles present in the ovary during the cycle 7 (Table 4A). During pregnancy, 70-115 (mean, 93) Type 3b follicles could be found in the ovary, while during the cycle their number varied between 113 and 165 (mean, 139). Thus, throughout pregnancy there were fewer follicles in the beginning stages of growth in the ovary than during the cycle.
48
Vol. 22
PEDERSEN AND PETERS
TABLE 4. The Kinetics of Type 3b Follicles at Different Times during Pregnancy and during the Estrus Cycle A. No. of Type 3b follicles present in one ovary
Day of pregnancy 2 4 8 10 12 17 Day of parturition (20 days after
B. D. Doubling C No. of times of Tr 't' t" £ II" I granulosa ansI nne 0 IC es ceUs in of 1yPe 3b startIn~ to Type 3b follIcles grow In follicles 24 hours hr.
70 115 95 95 95 90 140
79 SO 86 85 84 SO 76
173 175 188 186 184 175 186
20
139
81
178
19
11 16 12 12 12 12
vaginal plug) Mean values during
estrus cycle;
2. The transit time of Type 3b follicles was calculated after the doubling time had been determined, i.e., the time that it takes a cell population to double its number. 2 The doubling time (T D) of the cells in Type 3b follicles during pregnancy was calculated from the following formula 10 • 12 : LI
=
In 2 ( exp t.T;;-
-
) ( exp---r;; t2 In 2)
1
where t. and t2 represent the duration of the S-phase and of the G2-phase, respectively (t. and t2 were considered to be the same as in 28-day-old mice IO ). The doubling time of granulosa cells in
FIG. 5. Two types of corpora lutea are recognizable in ARG prepared during pregnancy after pulse labeling in the last prepregnancy estrus: the labeled corpora lutea of pregnancy (c.l.p.) and the unlabeled corpora lutea of previous cycles (c.l.). The ARG was prepared on Day 4 of pregnancy. H 3 TdR injected 8 days earlier. X 100.
January 1971
Type 3b follicles varied insignificantly from 79-86 hr. during pregnancy, and was found to be comparable to the doubling time of 81 hr. found during the cycle7 (Table 4B). The values were used to calculate the transit time of Type 3b follicles. The transit time varied little during the different stages of pregnancy and was similar to that during the estrus cycle (Table 4C). The number of follicles which start on their development as Type 3b follicles in a 24-hr. period any time during pregnancy can be calculated, considering that the number of Type 3b follicles present in the ovary does not vary during pregnancy (see above), that they grow continuously (see above), and that there is no significant degeneration of Type 3b follicles. lo The number which start development is equal to the number present in the ovary divided by the transit time. lO The number of follicles starting growth has been calculated for the different times during
I·
49
FOLLICLE GROWTH DURING PREGNANCY
pregnancy (Table 4D) and during the cycle. At any time during pregnancy, about 14 (11-16) follicles start to grow during a 24-hr. period, while during the cycle, about 19 (14-24) follicles leave the pool of small follicles and begin development every 24 hr. Thus, fewer follicles start to grow per time unit during pregnancy than during the cycle. It is of interest to note that a few hours after parturition the number of follicles that are stimulated to grow is again the same as during the cycle.
The Fate of the Prepregnancy Corpora Lutea The evolution of corpora lutea present in the ovary at the time of conception was followed in delayed ARG's prepared at different times of pregnancy after pulse labeling in the estrus before conception (Fig. 3). The labeling pattern in an ovary injected during estrus (Eo) is characteristic. l The freshly ruptured follicles (corpora
".
.~....
FIG. 6. The labeled corpus luteum of pregnancy shows heavily X 415.
<1t),
lightly
and unlabeled cells.
PEDERSEN AND PETERS
50
lutea of Eo) as well as the corpora lutea of previous cycles (Eo-I) remain free of labeled cells, while many granulosa cells in the medium and large follicles incorporate the label. If pregnancy is delayed until the next estrus occurs and an ARG of an ovary is prepared after conception has taken place, a clear distinction between the corpora lutea of pregnancy and those of previous cycles can be made (Fig. 5). The corpora lutea of pregnancy were formed from follicles which, at the time of injection, incorporated the label into many of their cells. Some of the granulosa cells divided before the follicle ruptured, while the theca cells apparently did not, or not as often as the granulosa cells. As a result, the corpora lutea of pregnancy, as seen in the delayed ARG, show heavily and lightly labeled as well as unlabeled cells (Fig. 6), while the corpora lutea of previous cycles are distinguished by a negative ARG. Labeled as well as unlabeled corpora lutea were regularly seen in all delayed ARG's prepared up to Day 12 of pregnancy. Some of the unlabeled prepregnancy corpora lutea were situated in the periphery of the ovary at the beginning of pregnancy. By Day 8 all the unlabeled corpora lutea were lying in the center of the organ. Before Day 12, prepregnancy corpora lutea were well defined; however, within a short time around that day, they had shrunk, disintegrated, and become reduced to islands and groups of luteal masses in the center of the organ. In ARG's prepared later than Day 12, only small remnants of prepregnancy corpora lutea could, at times, be recognized. DISCUSSION
These studies show that during pregnancy a continuous growth of follicles takes place. Throughout gestation, follicles are stimulated to leave the pool of nonproliferating small follicles and start on their development. This stimulation is
Vol. 22
not cyclic, but rather continuous, which causes follicles to start development at all times throughout the period of gestation. The stimulus necessary to start growth is apparently quantitatively different from that during the cycle, as fewer follicles begin their development per time unit during pregnancy. About 19 follicles begin development in a day during the cycle, while, during pregnancy, only 14 are stimulated to begin development in a 24-hr. period. However, those follicles that begin growth develop with the same speed as during the cycle. Thus, the "production line" of follicles continues uninterruptedly during pregnancy; only the number starting growth is smaller. The labeling pattern and the labeling index suggest that the follicles which had started development when pregnancy was initiated, as well as those which start growth during pregnancy, are not temporarily halted at any stage of their development nor at any stage of pregnancy. Those that begin growth continue until the hormonal balance prevailing during pregnancy does not support further development, and then they either degenerate or parts of their cell population are transformed and incorporated into the peripheral stroma. This supports Greenwald and Choudary's view4 that the pituitary, rather than the ovary, accounts for the reduced follicular activity between Days 12 and 14 of pregnancy, if "follicular activity" is defined as the ability to grow beyond a certain stage of development (Type 6). It must be presumed that the amount of gonadotropin necessary to further the growth of large follicles (beyond Type 5b and Type 6) is temporarily decreased during midpregnancy. This is mirrored in the reported sharp increase of atresia of antral follicles between Days 10 and 14 of pregnancy.4,13 A concomitant decrease in the number of large follicles4 during that period is apparently not caused by a failure to initiate follicle
•
L
/
l"!
FOLLICLE GROWTH DURING PREGNANCY
January 1971
,
!
51
growth, but, rather, by an inability to those follicles that began growth about support follicle growth beyond a certain the time that pregnancy started are thOse that will ovulate after the litter birth. All stage. Considering that the speed of follicle oocytes which started to grow in previous growth is comparable to that during the estrus cycles will have degenerated by cycle, and taking into consideration that the time gestation is ended, while those hormonal balance supports follicle growth that began development later will not only to a certain stage during the midpart have had time to reach preovulatory of pregnancy, one could speculate upon stages (Fig. 7). From the changes in the labeling patwhen oocytes, which come to ovulation at tern seen in the delayed ARG throughout the end of gestation, begin their growth. pregnancy, certain conclusions regarding Pedersen' determined the transit times for the formation of the peripheral stroma in the different follicle stages during the esthe ovary during gestation can be made. trus cycles (Table 5). As the speed of folThe immediate ARG showed cells constilicle growth is not altered significantly tuting the stroma free of label, while in during pregnancy, these transit times were the delayed ARG (6 days or more after used to estimate the approximate time pulse labeling) areas of labeled cells when the follicles that ovulated postparwithin the stroma became frequent. This tum began on their development. By addthat stroma continues to be formed implies ing the transit times, one realizes that during pregnancy and that it arises from follicles which did not develop beyond TABLE 5. Mean Transit Time of Follicles in a certain stage, a process known in the Ovaries of 3-Month-old Mice during the adult, nonpregnant ovary1, 12, 14 as well Cycle' in the infant ovary.5, 15 The delay of 6 3b 45a 5b67 days before labeled cells appear within Follicle type the stroma is explained by the fact that 178 129 62 25 14 51 Transit time (hr.) at the time of pulse labeling only healthy
TYPE 7 TYPE 6 TYPE Sb TYPE Sa
.., III C)
~
III
..,
TYPE 4
...J
U
::; ...J
0
lL
'.
TYPE 3b
i'
.
-CVCLUS~
.. <------PREGNANCV------
FIG. 7. Schematic representation of follicle growth during pregnancy. The height of the compartments of the different follicle stages represents the time that it takes for the follicle to grow through this compartment. The follicles that start growth within the first few days of pregnancy reach preovulatory size at the end of gestation.
52
PEDERSEN AND PETERS
Vol. 22
• follicles are proliferating and incorporate the label, while those already in degeneration do not. The autoradiographic studies confirm previous reports l3 • 16. 17 that the corpora lutea of previous cycles remain in the ovary for some time and undergo rapid degeneration about the 12th day of pregnancy, after which time only small traces remain in the center of the organ. SUMMARY
The growth and development of the ovary during pregnancy have been investigated. Autoradiographic studies show that there is a continuous initiation of follicle growth during pregnancy; i.e., the production line of follicles is uninterrupted. Fewer follicles start growth per time unit during pregnancy than during the estrus cycle. However, the speed with which they grow is the same during gestation and during the cycle. Follicle growth is not halted temporarily at any stage of development, or at any stage of pregnancy, but continues uninterruptedly until the hormone balance prevailing during pregnancy does not support further development. The follicles then either degenerate or part of their cells are incorporated into the stroma. With cell kinetic studies it was possible to estimate that those follicles that ovulate after the litter is born start their development at the beginning of gestation. The movement and disappearance of the prepregnancy corpora lutea could be followed in delayed autoradiographs. Acknowledgments. The authors wish to thank Brit Falck Madsen, Gurli Rasmussen, Annelise Hodel, Ingeborga Larsen, and Inge MtJller Jensen for their excellent technical assistance. REFERENCES 1. PETERS, H., AND LEVY, E. Cell dynamics of the ovarian cycle. J Reprod Fertilll:227, 1966.
2. PEDERSEN, T. Follicle growth in the immature mouse ovary. Acta Endocr 62:117, 1969. 3. DEMPSEY, E. W. Follicular growth rate and ovulation after various experimental procedures in guinea pig. Am J PhysioI120:126, 1937. 4. GREENWALD, G. S., AND CHOUDARY, J. B. Follicular development and induction of ovulation in the pregnant mouse. Endocrinology 84:1512, 1969. 5. PEDERSEN, T., AND PETERS, H. Proposal for a classification of oocytes and follicles in the mouse ovary. J Reprod Fertil17:555, 1968. 6. CRONE, M., PETERS, H., AND LEVY, E. The duration of the premeiotic DNA synthesis in mouse oocytes. Exp Cell Res 39:678, 1965. 7. PEDERSEN, T. Follicle kinetics in the 'ovary of the cyclic mouse. Acta Endocr. 64:304, 1970. 8. PILGRIM, C., AND MAURER, W. Autoradiographische Untersuchung fiber die Konstanz der DNS-Verdopplungs-Dauer der Zellarten von Maus und Ratte durch Doppelmarkierung mit 3H- und 14C-Thymidin. Exp Cell Res 37:183, 1965. 9. WIMBER, D. E., AND QUASTLER, H. A 14C- and 3H-thymidine double labeling technique in the study of cell proliferation in tradescantia root tips. Exp Cell Res 30:8, 1963. 10. PEDERSEN, T. Determination of follicle growth rate in the ovary of the immature mouse. J Reprod Fertil 21:81, 1970. 11. PETERS, H. Development of the mouse ovary from birth to maturity. Acta Endocr 62:98, 1969. 12. CLEAVER, J. E. The relationship between the duration of the S phase and the fraction of cells which incorporate 3-H-thymidine during exponential growths. Exp Cell Res 39:697,1965. 13. CHOUDARY, J. B., AND GREENWALD, G. 1. Ovarian activity in the intact or hypophysectomized pregnant mouse. Anat Rec 163:359, 1969. 14. DAWSON, A. B., AND MCCABE, M. J. Interstitial tissue of ovary in infantile and juvenile rats. • J MorphoI88:543, 1951. 15. BYSKov, A., PEDERSEN, T. AND PETERS, H. Proceedings of tjymposium on Development of the Ovary in Infancy, Birmingham, 1969. E. & S. Livingstone, Ltd. Edinburgh and London, In Press. 16. CHOUDARY, J: B., AND GREENWALD, G. S. Luteotropic complex of the mouse. Anat Rec 163:373, 1969. 17. DEANESLY, R. The corpora lutea of the mouse, with special reference to fat accumulation during the oestrus cycle. Proc Roy Soc [BioI] 106:578, 1930.
j'
FERTn.rrv AND STERn.1TY
Copyright
©
Printed in U.S.A.
1971 by 'The Williams & Wilkins Co.
FOLLICLE GROWTH AND CELL DYNAMICS IN THE MOUSE OVARY DURING PREGNANCY 1'-
TORBEN PEDERSEN
AND
HANNAH PETERS
The Finsen Laboratory, The Finsen Institute, Copenhagen, Denmark
It has recently become possible to fol- ments. They were mated, and the onset low the development and movements of of pregnancy was determined by the prescell populations in the ovary for a consid- ence of a vaginal plug (Day 0 of pregerable period of time in autoradiographs nancy). The average length of pregnancy after pulse labeling with tritiated thymi- is 19-20 days in this strain of mice. All mice were pulse-labeled by injectdine. 1 Moreover, the number of follicles that begin to grow at any time unit and ing intraperitoneally 100 JLC. of tritiated the speed of their development can be thymidine (H 3 TdR; Amersham Radiodetermined with follicle kinetic studies. 2 chemical Center, specific activity ranging The purpose of this investigation was to from 3.6-5.0 c./mM) either before or follow follicle growth and development during pregnancy. The animals were in the mouse ovary during pregnancy. killed at different time intervals after the Dempsey3 stated that pregnancy does not injection. Autoradiographs (ARG) were interfere with follicle growth in the guinea prepared of the serially sectioned (5 JL) . pig. Greenwald and Choudary4 reported a ovaries with K2 emulsion (Ilford), and extemporary decline in follicular activity in posed for 1-4 weeks at 4° C. They were the mouse ovary at the end of the second developed and then stained with hemaweek and a resumption of development at toxylin-eosin. At least 2 mice were used the beginning of the third week of gesta- for each determination. In the ARG of the ovaries, the labeling tion. In order to elucidate the complexity of follicle growth during pregnancy, the pattern as well as the labeling index of number of follicles which begin to develop the different structures was determined. during the different stages of pregnancy The distribution of labeled cells within a as well as their speed of growth was inves- structure (the follicle or the stroma reptigated. Furthermore, it seemed of interest resents the labeling pattern of the structo know whether those follicles which be- ture. The labeling index (LI) of a follicle gin to grow grow continuously or whether was defined as the number of labeled their development is held in abeyance at granulosa cells in percentage of the total any time. Finally, it seemed relevant to ob- number of cells; it was determined in the serve the fate of those developing follicles ARG 1 hr. after pulse labeling in that and corpora lutea which are already pres- cross-section of a follicle in which the ent in the ovary at the time that preg- nucleolus of the oocyte is seen. 2 nancy is initiated. These questions were To describe follicle growth a classificainvestigated in consecutive autoradio- tion of follicles based on the morphology graphs of ovaries of pregnant mice after and the number of granulosa cells was pulse labeling with tritiated thymidine. used5 (Fig. 1). Follicles of Type 3b were counted in MATERIALS AND METHODS every fifth section of the serially sectioned Sixty nulliparous Bagg mice, 3-4 ovary using the nucleolus of the oocyte as months old, were used in these experi- a marker. 42
'f
..
..
January 1971
(4~
~------
,
..... -
,,~t? ,// ¢V~ __ / 0" ",f:l~,/ TYPE
~ Iv'" /'
TYPE 3b
~ S~~ -' 'OJ
:
\
TYPE 3a
\,
0
~ 0 ~
;.
lSI
'.
"-'"
-'''-.
......
401-600 CEllS
61-100 CEllS >600 CEllS
~
'GI
\\'\,
.
TYPE 7
\\
\\\,
\.
e ! 20 CEllS
r, \
..J
(~s
--_" "P.J
TYPE 6
a~\ u..
~(/c '>0
21-60 CEllS
!
If)
~
G
oj,
---_
201-400 CEllS
~ 101-200 CEllS
TYPE 4
--____~yl'~
o
0 0
;j' ...." ./ ~ OV , /
/
50
--------__________ (OQG~ ....0
TYPE 5b
__ ---_7/;/'
~""\;;"O,',//
43
FOLLICLE GROWTH DURING PREGNANCY
TYPE 8
TYPE 2 'IS\' 'i?
\ "
\. ...
~
~
TYPE 1 @
FIG. 1. Classification of follicles
In order to determine the evolution of follicles and the fate of corpora lutea during pregnancy several experiments were performed.
The Growth of Follicles during Pregnancy Injection of HJTdR on Different days of Pregnancy. Twenty-two mice were injected with H 3 TdR on 1 of the following days of pregnancy: 2, 4, 8, 10, 12, 17, or 20. The animals were killed 1 hr. later and an ARG was prepared of their ovaries (immediate ARG). The labeling patterns of follicles and stroma, as well as the labeling index of follicles in different stages of development, were determined. The labeling pattern of follicles gave an indication of their growth: follicles containing labeled granulosa cells were proliferating while those without labeled cells were not. A change in the LI of a given follicle type indicated a change in the growth rate. 2 If, at any day, "healthy" medium or large follicles which on previous days had incorporated the label failed to do so, it was suggestive that the growth of that
follicle type had been held in abeyance. The labeling pattern of the stroma was observed to determine whether the stroma proliferated during pregnancy. Injection of 2 Different Doses of lfTdR on Day 12 of Pregnancy. In order to test the speed of follicle growth, the duration of the deoxyribonucleic acid (DNA) synthesis (S-phase) in granulosa cells was estimated on the 12th day of gestation. This was done indirectly, using the double labeling technic. s - 9 Two markedly differing doses of H 3TdR were used to produce heavily and lightly labeled cells in the ARG. Two mice were injected on Day 12 of pregnancy with 10 p.C. of H 3TdR and 1 hr. later with 100 p.C. of H 3 TdR. The animals were killed 45 min. after the last injection. For comparison, two 28-day-old mice which were not pregnant were treated in the same manner. The actual duration of the S-phase in granulosa cells had been previously determined in 28-day-old mice, using the method of labeled mitosis1o ; the results were used for comparison in this study. ARG's prepared after double labeling were exposed for 14 days. The number
44
PEDERSEN AND PETERS
of faintly labeled cells (up to 15 gr. over the nucleus) in percentage of heavily labeled cells was determined in the different follicles. Injection of lfTdR on the Day of the Vaginal Plug (Fig. 2). To follow the formation of the peripheral stroma, the following experiment was done. Twenty mice were mated, and, on the day of3 the vaginal plug (Day 0 of pregnancy), H TdR was injected. Autoradiographs were prepared at intervals of 1 hr. and 1, 2, 3, 4, 6, 8, 12, 17, and 20 days after pulse labeling. In the immediate ARG, proliferating and nonproliferating cell populations were determined. In autoradiographs prepared some days after the injection of H 3TdR (delayed ARG), the evolution of those structures that had incorporated the label at the beginning of pregnancy could be followed. A displacement of labeled cell groups became evident and, furthermore, cell divisions could be inferred if heavily labeled cells became replaced by lightly labeled cells. VAGINAL PLUG 3H-TdR
~----CORPUS LUTEUM OF PREGNANCY
LARGE FOLLICLE
0)-----GROWING FOLLICLE
@--------SMALL FOLLICLE
FIG. 2. Schematic illustration of label distribution in the ovary after pulse labeling on the day of the vaginal plug.
Vol. 22
r~
The Fate of the Prepregnancy Corpora Lutea
Injection of H 3 TdR before Conception (Fig. 3). In order to distinguish between the corpora lutea of pregnancy and those formed in cycles before conception and to follow their persistance during pregnancy, 16 mice were injected on the day of estrus (Eo) and mated 2 days later. Mice that showed a vaginal plug within 2 days were sacrificed on Days 2, 4, 6, 8, 12, 14, 18, and 20 of pregnancy for the preparation of an ARG of their ovaries. In such an experimental set-up, the corpora lutea of pregnancy and those of prepregnancy cycles can be distinguished, as the corpus luteum of pregnancy contain labeled cells, while the corpora lutea that were already in the ovary at the time of injection (corpora lutea of Eo and EO-I) remain free of the label. I RESULTS
Follicle Growth.during Pregnancy The immediate ARG obtained after injection of H3'J'dR on different days of pregnancy clearly showed the proliferating and nonproliferating cell systems in the ovary (Fig. 4). Cells of the medium and large follicles are proliferating while the cells of the stroma and corpora lutea are not. The number of small, Type 3a follicles that showed labeled granulosa cells varied from 20% at the beginning (Day 2) to 40% at the end (Day 20) of pregnancy (Table 1). However, all the medium and large follicles showed labeled granulosa cells after pulse labeling on the different days of pregnancy, indicating that DNA synthesis took place on all days tested. This suggests that all healthy medium and large follicles grow, and that none stops growth temporarily to resume it later in pregnancy. Follicles in degeneration as well as contracted follicles l l were free of labeled cells. Stroma cells were unlabeled in all immediate ARG's. However, in
\r
January 1971
45
FOLLICLE GROWTH DURING PREGNANCY
PREGNANCY
MALE IN CAGE
OESTRUS (Eo)
!
o
CORPUS LUTEUM OF EO-1
CORPUS LUTEUM OF EO
-~~-.
OESTRUS (E,)
o
o
.~ . ..;;~, .. ---® .
"
. ".~'''.'
. LARGE FOLLICLE
-
.
CORPUS LUTEUM OF PREGNANCY
@ GROWING FOLLICLE
@ SMALL FOLLICLE
LARGE FOLLICLE
@
-
GROWING FOLLICLE
FIG. 3. Schematic illustration of label distribution after pulse labeling at estrus and label dilution several days later at the time of fertilization.
ARG's prepared 6 days or later after pulse servations suggest that part of the periphlabeling on Day 0, areas of labeled cells eral stroma seen during pregnancy derives were seen in the peripheral stroma. These from contracted and transforming follicles. In order to define follicle growth in areas were numerous during midpreggreater detail, two factors are discussed: nancy and became fewer toward the end. (1) the speed with which they grow, in The cells in such areas were usually particular, the changing rate of growth lightly labeled, suggesting that they must during their development and (2) the have belonged to a cell population which, that begin to grow per number of follicles since pulse labeling, had divided and diunit time during pregnancy. luted the label. Delayed ARG's prepared The speed of growth of a follicle de6 or more days after pulse labeling in midpends upon the proliferation rate of its pregnancy also showed areas of -labeled granulosa cell population. This is detercells in the peripheral stroma. As only the the number of cells synthesizing mined by granulosa cells of medium and large folliDNA (expressed as the LI of the follicle) cles are proliferating during pregnancy, it and by the duration of the S-phase of the is inferred that the areas of labeled cells single granulosa cells. The LI in the varin the stroma are derived from cell groups ious types of growing follicles was deteroriginally belonging to follicles. These ob-
46
PEDERSEN AND PETERS
Vol. 22
;.-.
,
FIG. 4. Labeling pattern of an ovary on the 12th day of pregnancy 1 hr. after injection of H'TdR. Granulosa cells incorporated the label while the cells of corpora lutea and stroma remained unlabeled. X 170.
TABLE 1. Percentage of Small Follicles (Type 3a) Containing Labeled Granulosa Cells on Different Days of Pregnancy and during the Cycle in ARG Prepared 1 hr. after Pulse labeling No. of small
H'TdR injected
follicles (Type 3a) with labeled granulosa cells
On day of pregnancy 2 4 8 10 12 17 20 (day of litter birth) During estrus cycle
%
21
32 28 36 34 34
40 34 (mean value)
mined at different times during pregnancy and compared with those found in the nonpregnant mouse during a cycle7 (Table 2). The LI of large follicles was considerably higher than in medium follicles throughout pregnancy as well as during the estrus cycle. Considering the different
follicle types during pregnancy, it is noted that the LI in the Type 3b follicle is unaltered throughout pregnancy, while the LI of Types 4, 5a, and the large follicles tends to decrease temporarily at midpregnancy; the differences in the values are, however, statistically not significant at a 5% level. The LI in comparable follicle types during pregnancy and during the cycle are similar, indicating that the number of granulosa cells synthesizing DNA in comparable follicles is similar during pregnancy and during the cycle. The duration of the S-phase was investigated indirectly for medium and large follicles on the 12th day of pregnancy. In ARG's prepared after 2 differing doses of H 3 TdR (double labeling experiment) the percentage of faintly labeled cells in relation to heavily labeled cells was determined for follicles in 3-month-old pregnant mice as well as in 28-day-old virgin mice. A comparison of the percent of lightly labeled cells in the two groups of mice permitted a comparison of the dura-
..
.....
Jarwary 1971
47
FOLLICLE GROWTH DURING PREGNANCY
~
TABLE 2. Labeling Index of Follicles on Different Days of PreglUlncy and during the Estrus Cycle
...
Large foDicles
Medium foDicles
""
Mean
~
%
":,
-. -< "-
Day of pregnancy 2 4 8 10 12 17 20 (day oflitter
Type 5a
Type 4
Type 3b
S.E.
Mean
Mean
S.E.
S.E.
Mean
S.E.
'1 ~
S.E.
Mean
S.E.
%
9.2 9.1 8.4 8.6 8.5 9.1 9.6
0.9 0.8 1.2 1.2 0.8 0.7 0.9
7.7 7.5 6.0 5.6 7.1 6.0 7.4
0.9 0.8 0.9 0.7 0.5 0.6 0.7
15.6 15.2 16.1 11.5 11.7 11.6 20.9
3.2 2.3 2.7 3.4 1.5 1.5 3.0
42.1 25.8 23.8 29.3 27.9 35.7 42.9
3.4 2.7 2.5 2.1 2.0 1.6 3.0
22.2 25.0 44.1* 27.7 24.2 43.6*
0.8 4.4
9.3
0.2
7.3
0.2
15.3
0.5
35.8
1.0
35.2
1.4
birth)
Mean LI during estrus cycle
Mean
Type 7
%
%
%
%
Type 6
Type 5b
4.0 2.8
22.3 20.9 15.9 20.0* 16.4 16.8 22.2
1.7 8.7 2.4
28.0
1.5
1.9 2.2 8.7
* Based on one detennination only.
TABLE 3. Percentage of Faintly Labeled Cells in Follicles after Injection of Two Different Doses (10 ,.c. and 100 ,.c.) of lfTdR on the 12th Day of PreglUlncy and in 28-Day-Old Virgin Mice Medium foDicles
Large foDicle.
Type 3b Type 4 Type 5a Type 5b Type 6 %
12th day of pregnancy \3month-old mice) 2S-Day_old-virgin mice
13.5
14.5
13.9
18.5
17.7
14.7
13.0
13.9
16.8
16.5
9.8
11.2
hr. Duration of S-phase in granulosa cells determined in 28-day-old
10.3
6.8
6.8
virgin mice7
tion of the S-phase, as the following relationship exists9 : t.
Nh Nf
=
ta
where Nh is the number of heavily labeled cells, Nt is the number of faintly labeled cells, t. is the duration of the Sphase, and ta is the interval between injections. The values for the percentage of faintly labeled cells in comparable follicle types
in the two groups of mice were obtained (Table 3). They were not different at the 5% level, suggesting that the DNA synthesis time is comparable and that no large variation in the duration of DNA synthesis time in comparable follicles has occurred on the 12th day of pregnancy. In order to determine the number of follicles that begin to grow during a 24-hr. period, two factors have to be known: (1) the number of Type 3b follicles present in the ovary and (2) the transit time,2 i.e., the time that it takes a 3b follicle to grow from one with 21 to one with 60 granulosa cells on its largest cross section (Fig. 1). 1. The total number of Type 3b follicles present in the ovary of mice at different stages of pregnancy was determined and compared with the number of Type 3b follicles present in the ovary during the cycle 7 (Table 4A). During pregnancy, 70-115 (mean, 93) Type 3b follicles could be found in the ovary, while during the cycle their number varied between 113 and 165 (mean, 139). Thus, throughout pregnancy there were fewer follicles in the beginning stages of growth in the ovary than during the cycle.
48
Vol. 22
PEDERSEN AND PETERS
TABLE 4. The Kinetics of Type 3b Follicles at Different Times during Pregnancy and during the Estrus Cycle A. No. of Type 3b follicles present in one ovary
Day of pregnancy 2 4 8 10 12 17 Day of parturition (20 days after
B. D. Doubling C No. of times of Tr 't' t" £ II" I granulosa ansI nne 0 IC es ceUs in of 1yPe 3b startIn~ to Type 3b follIcles grow In follicles 24 hours hr.
70 115 95 95 95 90 140
79 SO 86 85 84 SO 76
173 175 188 186 184 175 186
20
139
81
178
19
11 16 12 12 12 12
vaginal plug) Mean values during
estrus cycle;
2. The transit time of Type 3b follicles was calculated after the doubling time had been determined, i.e., the time that it takes a cell population to double its number. 2 The doubling time (T D) of the cells in Type 3b follicles during pregnancy was calculated from the following formula 10 • 12 : LI
=
In 2 ( exp t.T;;-
-
) ( exp---r;; t2 In 2)
1
where t. and t2 represent the duration of the S-phase and of the G2-phase, respectively (t. and t2 were considered to be the same as in 28-day-old mice IO ). The doubling time of granulosa cells in
FIG. 5. Two types of corpora lutea are recognizable in ARG prepared during pregnancy after pulse labeling in the last prepregnancy estrus: the labeled corpora lutea of pregnancy (c.l.p.) and the unlabeled corpora lutea of previous cycles (c.l.). The ARG was prepared on Day 4 of pregnancy. H 3 TdR injected 8 days earlier. X 100.
January 1971
Type 3b follicles varied insignificantly from 79-86 hr. during pregnancy, and was found to be comparable to the doubling time of 81 hr. found during the cycle7 (Table 4B). The values were used to calculate the transit time of Type 3b follicles. The transit time varied little during the different stages of pregnancy and was similar to that during the estrus cycle (Table 4C). The number of follicles which start on their development as Type 3b follicles in a 24-hr. period any time during pregnancy can be calculated, considering that the number of Type 3b follicles present in the ovary does not vary during pregnancy (see above), that they grow continuously (see above), and that there is no significant degeneration of Type 3b follicles. lo The number which start development is equal to the number present in the ovary divided by the transit time. lO The number of follicles starting growth has been calculated for the different times during
I·
49
FOLLICLE GROWTH DURING PREGNANCY
pregnancy (Table 4D) and during the cycle. At any time during pregnancy, about 14 (11-16) follicles start to grow during a 24-hr. period, while during the cycle, about 19 (14-24) follicles leave the pool of small follicles and begin development every 24 hr. Thus, fewer follicles start to grow per time unit during pregnancy than during the cycle. It is of interest to note that a few hours after parturition the number of follicles that are stimulated to grow is again the same as during the cycle.
The Fate of the Prepregnancy Corpora Lutea The evolution of corpora lutea present in the ovary at the time of conception was followed in delayed ARG's prepared at different times of pregnancy after pulse labeling in the estrus before conception (Fig. 3). The labeling pattern in an ovary injected during estrus (Eo) is characteristic. l The freshly ruptured follicles (corpora
".
.~....
FIG. 6. The labeled corpus luteum of pregnancy shows heavily X 415.
<1t),
lightly
and unlabeled cells.
PEDERSEN AND PETERS
50
lutea of Eo) as well as the corpora lutea of previous cycles (Eo-I) remain free of labeled cells, while many granulosa cells in the medium and large follicles incorporate the label. If pregnancy is delayed until the next estrus occurs and an ARG of an ovary is prepared after conception has taken place, a clear distinction between the corpora lutea of pregnancy and those of previous cycles can be made (Fig. 5). The corpora lutea of pregnancy were formed from follicles which, at the time of injection, incorporated the label into many of their cells. Some of the granulosa cells divided before the follicle ruptured, while the theca cells apparently did not, or not as often as the granulosa cells. As a result, the corpora lutea of pregnancy, as seen in the delayed ARG, show heavily and lightly labeled as well as unlabeled cells (Fig. 6), while the corpora lutea of previous cycles are distinguished by a negative ARG. Labeled as well as unlabeled corpora lutea were regularly seen in all delayed ARG's prepared up to Day 12 of pregnancy. Some of the unlabeled prepregnancy corpora lutea were situated in the periphery of the ovary at the beginning of pregnancy. By Day 8 all the unlabeled corpora lutea were lying in the center of the organ. Before Day 12, prepregnancy corpora lutea were well defined; however, within a short time around that day, they had shrunk, disintegrated, and become reduced to islands and groups of luteal masses in the center of the organ. In ARG's prepared later than Day 12, only small remnants of prepregnancy corpora lutea could, at times, be recognized. DISCUSSION
These studies show that during pregnancy a continuous growth of follicles takes place. Throughout gestation, follicles are stimulated to leave the pool of nonproliferating small follicles and start on their development. This stimulation is
Vol. 22
not cyclic, but rather continuous, which causes follicles to start development at all times throughout the period of gestation. The stimulus necessary to start growth is apparently quantitatively different from that during the cycle, as fewer follicles begin their development per time unit during pregnancy. About 19 follicles begin development in a day during the cycle, while, during pregnancy, only 14 are stimulated to begin development in a 24-hr. period. However, those follicles that begin growth develop with the same speed as during the cycle. Thus, the "production line" of follicles continues uninterruptedly during pregnancy; only the number starting growth is smaller. The labeling pattern and the labeling index suggest that the follicles which had started development when pregnancy was initiated, as well as those which start growth during pregnancy, are not temporarily halted at any stage of their development nor at any stage of pregnancy. Those that begin growth continue until the hormonal balance prevailing during pregnancy does not support further development, and then they either degenerate or parts of their cell population are transformed and incorporated into the peripheral stroma. This supports Greenwald and Choudary's view4 that the pituitary, rather than the ovary, accounts for the reduced follicular activity between Days 12 and 14 of pregnancy, if "follicular activity" is defined as the ability to grow beyond a certain stage of development (Type 6). It must be presumed that the amount of gonadotropin necessary to further the growth of large follicles (beyond Type 5b and Type 6) is temporarily decreased during midpregnancy. This is mirrored in the reported sharp increase of atresia of antral follicles between Days 10 and 14 of pregnancy.4,13 A concomitant decrease in the number of large follicles4 during that period is apparently not caused by a failure to initiate follicle
•
L
/
l"!
FOLLICLE GROWTH DURING PREGNANCY
January 1971
,
!
51
growth, but, rather, by an inability to those follicles that began growth about support follicle growth beyond a certain the time that pregnancy started are thOse that will ovulate after the litter birth. All stage. Considering that the speed of follicle oocytes which started to grow in previous growth is comparable to that during the estrus cycles will have degenerated by cycle, and taking into consideration that the time gestation is ended, while those hormonal balance supports follicle growth that began development later will not only to a certain stage during the midpart have had time to reach preovulatory of pregnancy, one could speculate upon stages (Fig. 7). From the changes in the labeling patwhen oocytes, which come to ovulation at tern seen in the delayed ARG throughout the end of gestation, begin their growth. pregnancy, certain conclusions regarding Pedersen' determined the transit times for the formation of the peripheral stroma in the different follicle stages during the esthe ovary during gestation can be made. trus cycles (Table 5). As the speed of folThe immediate ARG showed cells constilicle growth is not altered significantly tuting the stroma free of label, while in during pregnancy, these transit times were the delayed ARG (6 days or more after used to estimate the approximate time pulse labeling) areas of labeled cells when the follicles that ovulated postparwithin the stroma became frequent. This tum began on their development. By addthat stroma continues to be formed implies ing the transit times, one realizes that during pregnancy and that it arises from follicles which did not develop beyond TABLE 5. Mean Transit Time of Follicles in a certain stage, a process known in the Ovaries of 3-Month-old Mice during the adult, nonpregnant ovary1, 12, 14 as well Cycle' in the infant ovary.5, 15 The delay of 6 3b 45a 5b67 days before labeled cells appear within Follicle type the stroma is explained by the fact that 178 129 62 25 14 51 Transit time (hr.) at the time of pulse labeling only healthy
TYPE 7 TYPE 6 TYPE Sb TYPE Sa
.., III C)
~
III
..,
TYPE 4
...J
U
::; ...J
0
lL
'.
TYPE 3b
i'
.
-CVCLUS~
.. <------PREGNANCV------
FIG. 7. Schematic representation of follicle growth during pregnancy. The height of the compartments of the different follicle stages represents the time that it takes for the follicle to grow through this compartment. The follicles that start growth within the first few days of pregnancy reach preovulatory size at the end of gestation.
52
PEDERSEN AND PETERS
Vol. 22
• follicles are proliferating and incorporate the label, while those already in degeneration do not. The autoradiographic studies confirm previous reports l3 • 16. 17 that the corpora lutea of previous cycles remain in the ovary for some time and undergo rapid degeneration about the 12th day of pregnancy, after which time only small traces remain in the center of the organ. SUMMARY
The growth and development of the ovary during pregnancy have been investigated. Autoradiographic studies show that there is a continuous initiation of follicle growth during pregnancy; i.e., the production line of follicles is uninterrupted. Fewer follicles start growth per time unit during pregnancy than during the estrus cycle. However, the speed with which they grow is the same during gestation and during the cycle. Follicle growth is not halted temporarily at any stage of development, or at any stage of pregnancy, but continues uninterruptedly until the hormone balance prevailing during pregnancy does not support further development. The follicles then either degenerate or part of their cells are incorporated into the stroma. With cell kinetic studies it was possible to estimate that those follicles that ovulate after the litter is born start their development at the beginning of gestation. The movement and disappearance of the prepregnancy corpora lutea could be followed in delayed autoradiographs. Acknowledgments. The authors wish to thank Brit Falck Madsen, Gurli Rasmussen, Annelise Hodel, Ingeborga Larsen, and Inge MtJller Jensen for their excellent technical assistance. REFERENCES 1. PETERS, H., AND LEVY, E. Cell dynamics of the ovarian cycle. J Reprod Fertilll:227, 1966.
2. PEDERSEN, T. Follicle growth in the immature mouse ovary. Acta Endocr 62:117, 1969. 3. DEMPSEY, E. W. Follicular growth rate and ovulation after various experimental procedures in guinea pig. Am J PhysioI120:126, 1937. 4. GREENWALD, G. S., AND CHOUDARY, J. B. Follicular development and induction of ovulation in the pregnant mouse. Endocrinology 84:1512, 1969. 5. PEDERSEN, T., AND PETERS, H. Proposal for a classification of oocytes and follicles in the mouse ovary. J Reprod Fertil17:555, 1968. 6. CRONE, M., PETERS, H., AND LEVY, E. The duration of the premeiotic DNA synthesis in mouse oocytes. Exp Cell Res 39:678, 1965. 7. PEDERSEN, T. Follicle kinetics in the 'ovary of the cyclic mouse. Acta Endocr. 64:304, 1970. 8. PILGRIM, C., AND MAURER, W. Autoradiographische Untersuchung fiber die Konstanz der DNS-Verdopplungs-Dauer der Zellarten von Maus und Ratte durch Doppelmarkierung mit 3H- und 14C-Thymidin. Exp Cell Res 37:183, 1965. 9. WIMBER, D. E., AND QUASTLER, H. A 14C- and 3H-thymidine double labeling technique in the study of cell proliferation in tradescantia root tips. Exp Cell Res 30:8, 1963. 10. PEDERSEN, T. Determination of follicle growth rate in the ovary of the immature mouse. J Reprod Fertil 21:81, 1970. 11. PETERS, H. Development of the mouse ovary from birth to maturity. Acta Endocr 62:98, 1969. 12. CLEAVER, J. E. The relationship between the duration of the S phase and the fraction of cells which incorporate 3-H-thymidine during exponential growths. Exp Cell Res 39:697,1965. 13. CHOUDARY, J. B., AND GREENWALD, G. 1. Ovarian activity in the intact or hypophysectomized pregnant mouse. Anat Rec 163:359, 1969. 14. DAWSON, A. B., AND MCCABE, M. J. Interstitial tissue of ovary in infantile and juvenile rats. • J MorphoI88:543, 1951. 15. BYSKov, A., PEDERSEN, T. AND PETERS, H. Proceedings of tjymposium on Development of the Ovary in Infancy, Birmingham, 1969. E. & S. Livingstone, Ltd. Edinburgh and London, In Press. 16. CHOUDARY, J: B., AND GREENWALD, G. S. Luteotropic complex of the mouse. Anat Rec 163:373, 1969. 17. DEANESLY, R. The corpora lutea of the mouse, with special reference to fat accumulation during the oestrus cycle. Proc Roy Soc [BioI] 106:578, 1930.
j'