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Atresia of the dominant ovarian follicle in rhesus monkeys is detected within 24 hours of estradiol treatment.

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American Journal of Primatology 18:237-243 (1989)
Atresia of the Dominant Ovarian Follicle in
Rhesus Monkeys Is Detected Within
24 Hours of Estradiol Treatment
REINHOLD J. HUTZ1, GAIL S. KRUEGER2, PATRICIA M. MORGAN2,
DONALD J. DIERSCHKE2, AND RICHARD C. WOLF2
'Department ofBiological Sciences, University of Wisconsin, Milwaukee and 'Wisconsin
Regional Primate Research Center, Madison
Following treatment with estradiol-17P (E2) on day 6 of the menstrual
cycle, degenerative alterations in the microenvironment of the dominant
follicle (DF) (follicular fluid [FFI, granulosa cells IGCI, and oocyte) are
readily apparent on day 10, or 96 h after E2 administration. The present
study was designed to determine how early such changes could be detected
and which indices of atresia were observed first. The DF was identified
during laparoscopy on day 5 or 6 of the cycle, and four capsules containing
crystalline E2 were inserted S.C.for 24 h. Contents of the DF were aspirated a t 24,48, and 72 h following initiation of E2 treatment. General size
and appearance of the DF did not change distinctly with E2 treatment;
however, by 48 h FF viscosity was increased markedly. GC viability was
not altered with treatment. FF concentrations of estrogen (E) were dramatically reduced at 24 h. These differences were maintained a t 48 h and
at 72 h. E accumulation by cultured GC was significantly reduced by >
eightfold. There appeared a similar trend for reduced progesterone (PI in
FF and decreased P production by GC in vitro. These results demonstrate
that degenerative alterations in the DF indicative of atresia can be detected as early as 24 h after initiation of E2 treatment; the index of atresia
appearing earliest is a reduction in FF concentrations of E, and the first
morphological changes in the DF can be observed 24 h later. This study
indicates that biochemical alterations precede morphologic changes with
E2-induced atresia, and should allow us to begin to determine the earliest
events and putative initiation sites of atresia.
Key words: Macaca mulatta, degeneration, steroids
INTRODUCTION
We previously developed a model for the analysis of ovarian follicular atresia
in rhesus monkeys [Dierschke et al., 1985; Hutz et al., 1986al. Administration of
estradiol-17P (E2)via Silastic capsules S.C.on day (d) 6 of the menstrual cycle for
a 24-hour (h) period elicited degenerative changes in the dominant preovulatory
follicle (DF) indicative of atresia; these included a diminution in granulosa cell
Received for publication December 12, 1988; revision accepted April 21, 1989.
Address reprint requests to Dr. Reinhold J. Hutz, Department of Biological Sciences, University of
Wisconsin-Milwaukee, P.O. Box 413, Milwaukee, WI 53201.
0 1989 Alan R. Liss. Inc.
238 I Hutz et al.
(GC) viability and steroidogenic capability in vitro, in follicular fluid (FF) concentrations of estrogen (E) and progesterone (P), and in viability of the oocyte. These
alterations were observed a t 96 h following initiation of E2 treatment. The present
study was designed to determine how quickly morphologic and endocrinologic
changes could be detected following treatment, and which indices of atresia are
manifested first.
METHODS
Animals
Animal care and housing, blood sampling, and surgical procedures were as
described previously [Clark et al., 1978; Hutz et al., 19851. Forty-one adult rhesus
monkeys that exhibited cycles (24-32 d) and luteal phases (>lo d) of normal
length were used. Femoral or saphenous venous samples (3 ml) were taken daily
from d 1of the cycle (first day of menses) to the d of aspiration of follicular contents;
sera were assayed for luteinizing hormone (LH) and E. Blood samples (3 ml) were
taken twice weekly in the luteal phase for P assay.
Aspiration and Cell Culture
The DF was identified and mean diameter measured at laparoscopy [Dierschke
& Clark, 19761 on d 5 or 6 of the menstrual cycle. Silastic capsules containing
crystalline E2 [Dierschke et al., 19851 were inserted S.C.on d 6 and removed 24 h
later. Follicular contents were aspirated as previously described on d 7, 8, or 9 of
the cycle [Hutz et al., 1986al. FF was diluted and frozen until assayed for E and P.
The number of GC aspirated (determined by counting in a hemocytometer) averaged 5.93 ? 0.82 x lo5 (41) (R
1 SEM, [n]; range, 1.75 x lo4-1.98 x lo6). GC
number was not altered by E2 treatment on any cycle day. The mean percentage of
GC classified as viable (those excluding trypan blue vital dye, 0.2%; Sigma, St.
Louis, MO) was 44.0 -+ 2.6% (n=39); (range, 18.0-94.0%). GC were plated at a
density of 5 x lo4 viable cells per 0.5 ml aliquot of Ham’s F12: Dulbecco’s modified
Eagle’s medium (1:l) [Sanders and Midgley, 19831, containing human folliclestimulating hormone (hFSH, 100 ng/ml) for 72 h. Estimation of E and P accumulation in culture was expressed as ng steroidi0.5 ml aliquot containing 5 x lo4
viable GC a t the start of culture per 72 h.
*
Radioimmunoassays
Concentrations of LH, E, and P in serum and of steroids in FF and culture
medium were validated and quantitated according to previously published methods [Clark et al., 1978,1979; Hutz et al., 1986al. Occurrence of the serum LH peak
was noted with reference to d 8 of the cycle, since an elevation in LH or a “rebound”
phenomenon occurs on this day in untreated monkeys in response to removal of E2
capsules [Dierschke et al., 19851.
Statistical Analysis
Amounts of circulating hormones were quantitated by integrating and digitizing the areas under hormone curves (area computer program courtesy of the
Wisconsin Regional Primate Research Center) [Hutz et al., 19851; data were normalized to d 9 of the cycle, as prior to this time P was typically not detectable. Some
data in Table I were incomplete due to inadequate GC recovery. Data from controls
and treatment were compared by Student’s t test or rank-sum test within a single
day of cycle (follicle diameter, granulosa cell viability), or by two-way analysis of
variance followed by Student-Newman-Keul’s test (Table I); and by Chi-square
contingency tables or Fisher exact-probability test (morphology of DF and FF)
7
Control
8
Treated
Control
9
Treated
46.6
-+
18.0 (6)b
5.10 f 1.49 (3)"
No
22.17 f 9.67 (2)"
No
No
36.00 -+ 0.88
-
-
Yes
26.97 f 4.95 (8)" 49.14 f 13.20 (7)"
31.91 f 5.79 (8)"
f 13.18 (5)"
~
5.06 t 0.68 (4Ib* 62.43 k 2.40 ( 2 ) " ~43.76
409.0 f 89.6 (7)"
62.6
f
16.7
(6)b
Equivocal
Yes
32.25 f 8.54 (6)" 43.68 f 6.47 (5)"
52.39 -+ 23.89 (4)" 70.13 k 23.86 (6)"
110.6 f 27.2 (4)b
f 139.7(6)"xb
~
728.3 f 129.6 ( 5 ) " ~835.6
745.3 f 30.2 (8Ib 533.9 f 90.9 (8)" 826.0 -t- 73.0 (7)b
30.3 f 6.5
(5)"
123.7 f 26.1 (7Ib 1193.1 f 416.9 (8)" 289.9 f 59.1 (6)"sb* 2237.5 f 562.8 (5)"
Treated
'Values are k 1 SEM (n).
2Digitized areas under hormone curves (estrogen, E; progesterone, P); values are relative units for integrated areas under curves using x-axis as the base.
3This group showed dichotomous E concentrations that were significantly different and was therefore subdivided.
a,b,eDifferencesin superscripts within rows designate significant (P < 0.05).
*P < 0.10 with respect to control.
x
Granulosa cell
accumulation
(ng/m1/50,000
viable cells/72 h)
Occurrence of
serum LH peak
on day 8
Estrogen
529.5 f 13.5 (3)"
Serum (area)'
Follicular fluid
1995.5 f 612.3(5)"
(ng/ml)
Granulosa cell
accumulation
(ng/m1/50,000
viable cells/72 h)
Progesterone
Serum (area)'
29.40 f 5.19 (6)"
Follicular fluid
30.40 f 10.82(5)"
(ngiml)
Control
Day of cycle
TABLE I. Endocrinologic Characteristics of Ovarian Follicles Examined 1,2, or 3 Days After Rhesus Monkeys Were Treated With
Estradiol-17B S.C. for 24 h on d 6 of the Menstrual Cycle'
240 / Hutz et al.
TABLE 11. Morphologic Characteristics of Ovarian Follicles Examined 1, 2, or 3 Days
After Rhesus Monkeys Were Treated With EstradioLl'7PS.C. for 24 h on d 6 of the
Menstrual Cycle'
Day of cycle
8
7
Control
Treated
Control
9
Treated
Control
Treated
Follicle
diameter
5.5 2 0.7 (6Iajb 4.9 i 0.3 (8)a 5.6 i 0.3
5.9 i 0.3 (71b 5.8 2 0.7 (61a,b 5.2 2 0.3 (6)a,b
(mm)
Follicle appearance
6/6a
818a
71Ta
6/6a
3/6*
(3 translucent)
elearltotal)
Follicular fluid
appearance
(no. normal1
6/6a
818a
818a
217b
616a
416a
( 5 viscous)
(2 viscous)
total)
Granulosa cell
viability (%) 29.0 i 6.8 (5Ia 42.9 i 3.8 (6Iaxb 51.0 i 6.8 (8Ib 44.8 i 5.4 (71azb 34.5 i 6.4 (6)a,b 44.3 i 5.7 (6)a,b
'Values are 2 1 SEM (n).
",bDifferences in superscripts within rows designate significance ( P < 0.05).
*P < 0.10.
[Zar, 19741. Steroid data were transformed (fi
or log x), as were percentage data
(arcsin 6 1 , prior to analysis. P < 0.05 was considered to be significant.
RESULTS
Morphologic alteration in the DF of monkeys treated with E2 on d 6 of the
menstrual cycle was evident by 48-72 h following Ez administration-this was
initially manifest as increased viscosity of FF at 48 h (Table 11); greater suction
was required for successful aspiration than controls, and less bleeding was seen.
This change was followed by apparent increased opacity of the DF in treated
animals (72 h; Table 11). Follicle diameter was similar on all cycle days (Table 11).
GC viability was significantly higher in d 8 controls vs. d 7 controls (Table 11).
There was no evidence of luteinization of GC in culture by either morphology or by
staining with oil red "0,"which is specific for lipids (Sigma, St. Louis, MO). Viability of plated GC was maintained near 100% throughout culture.
Endocrinologic manifestations of atresia were evident earlier than were morphologic indices. FF concentrations of E and P were markedly reduced within 24 h
of treatment (Table I). This trend in reduced E amounts continued for d 8 and 9, but
not for P, which remained at a plateau; however, FF P was reduced a t d 10 (previous results) [Hutz et al., 1986al. GC accumulation of E and P was reduced (Table
I), but only significantly for E on d 8. As expected, areas under the E curve were
elevated, as greater circulating amounts of E were characteristic of E2 treatment
(Table I). Areas under the luteal P curves did not change significantly with treatment (Table I). Augmented amounts of peripheral LH were observed in treated
animals on d 8 of the cycle, when aspiration was performed either d 8 or 9.
DISCUSSION
This study describes the temporal sequence of atretic changes occurring within
the DF of rhesus monkeys following systemic administration of E2. We previously
Time Course of Ovarian Follicle Atresia / 241
demonstrated that dramatic transformations in the intrafollicular milieu are evident by 96 h following treatment [Hutz et al., 1986al; the changes included reductions in FF amounts of steroids, in steroidogenic capacity of GC in culture, and
in viability of GC and oocytes. We now find that the morphologic modifications are
manifested 48 h earlier, and the biochemical changes a full 72 h earlier, or within
24 h of administering Ez.
The apparent increase in viscosity of the FF and opacity of the DF a t 48 and 72
h, respectively, were also observed a t 96 h [Hutz et al., 1986al. The low GC viability extant in control aspirates a t d 7 is difficult to explain; it may have been due
to the small size of follicles and relative difficulty of aspiration on this cycle day,
which resulted in a large variance in this group; there were no other differences
among GC viabilities, regardless of cycle day or treatment, and these values were
similar to those calculated for d 10 [Hutz et al., 1986al. The apparent lack of effect
of Ez on GC viability to d 9 may be due to the possibility that GC viability as
estimated by trypan blue exclusion is not affected t o a significant extent until 96
h after E2 treatment, manifesting an “all-or-none” rather than a graded response.
FF amounts of steroids in control follicles and their diminution with treatment
were comparable to our previous results on d 10. Interestingly, E production by GC
in vitro in some cultures was far greater than we reported previously; however,
this may, in part, be attributed to inter-assay variation (-17%), and the fact that
100 ng/ml hFSH were added to all cultures in the present study.
Due to the large variation in circulating E on d 9, there was no significant
increase in area under the curve with treatment, which would have been expected
due to the implanted capsules containing E2. Unexpectedly, there were no reductions in areas under the P curves with Ez treatment on either d 8, 9, or 10 of the
cycle; this is in contrast to what we have observed for P secretion after treatment
with E2 alone on d 6 [Dierschke et al., 19851, and different from limited preliminary results of the combination of treatment and aspiration on d 10 [Hutz et al.,
1986133;these latter observations await further corroboration. However, our results
regarding P secretion are quite similar to what we have shown following aspiration alone on d 10 [Hutz et al., 1987al. It is plausible that the mere act of aspiration
removed enough steroidogenically active GC [Kreitman et al., 19811on d 8,9, or 10
such that P production was lowered in all treatments equally, regardless of additional treatmentb). Also, variability in areas under hormone curves within groups
was sufficient to obscure any obvious differences between groups [Hutz et al.,
1987al.
The increase in circulating LH apparent on d 8 of the cycle, when aspiration
was performed on either d 8 or 9, was due to the rebound phenomenon characteristically observed on this day [Dierschke et al., 1985, 19871. We have in these
papers demonstrated conclusively that this phenomenon was not due to the endogenous LH surge, but was rather a consequence of E2-capsule removal.
The locus of E i s atretogenic effect(s) is controversial [Zeleznik et al., 1985;
Dierschke et al., 19873. From the collective evidence we have accrued, it appears
that E2 does suppress circulating FSH, presumably by an action a t the hypothalamic-pituitary level, but that E2 can exert a direct effect at the level of the ovary
[Hutz et al., 198733, 1987c, 1988, 19891. Reduced FSH or a direct action of Ez may
adversely affect aromatase activity [Hutz et al., 1987~1,thereby reducing GC production of Ez and hence increasing androgens and the likelihood of follicular
atresia.
242 I Hutz et al.
CONCLUSIONS
1. We have, for the first time, delineated systematically a time course for the
onset of estradiol-induced atresia of ovarian follicles in rhesus monkeys.
2. Endocrinologic alterations such as reduced follicular fluid steroids and
granulosa cell steroidogenesis are evident as early as 24 hours following estradiol
treatment, while morphologic indices of atresia are first observed 1 day later.
ACKNOWLEDGMENTS
The authors wish t o thank Ms. Maureen Durning for data acquisition and
analysis; Dr. S. Raiti of the National Hormone and Pituitary Program for the
hFSH; and Ms. Alnita Allen and Ms. Dorothy Perry for the faithful preparation of
this manuscript. This study was conducted in accordance with the “Guide for the
Care and Use of Laboratory Animals,” NIH Publication No. 85-23 and Public Law
89-544, “The Animal Welfare Act,” August 24, 1966, and its amendments. A preliminary report of this investigation was presented a t the 10th Annual Meeting of
the American Society of Primatologists, Madison, WI, June 13-16, 1987. This
research was supported by NIH Grants HD-17370 and RR-00167. Publication 28034 of the Wisconsin Regional Primate Research Center. Patricia M. Morgan’s
present address is Department of Physiology, University College Galway, Galway,
Ireland.
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