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Original Studies |
Division of Reproductive Sciences, Oregon Regional Primate Research Center (D.M.D., R.L.S.), Beaverton, Oregon 97006; the Division of Reproductive Biology and Medicine, University of California (D.R.S.), Davis, California 95616; and the Department of Physiology and Pharmacology, Oregon Health Sciences University (R.L.S.), Portland, Oregon 97201
Address all correspondence and requests for reprints to: Dr. Richard L. Stouffer, Division of Reproductive Sciences, Oregon Regional Primate Research Center, 505 NW 185th Avenue, Beaverton, Oregon 97006.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies have examined the roles of LH pulse amplitude and frequency in the maintenance of the primate corpus luteum. In the macaque, frequent LH pulses of lower amplitude were measured in serum early in the luteal phase, but by days 10–11 of the luteal phase, higher amplitude LH pulses were measured only three times daily (4). Monkeys with radiofrequency lesions to ablate endogenous GnRH production and subsequent pulsatile GnRH infusion were used to investigate the requirements for GnRH (and, presumably, LH) pulse frequency to maintain primate luteal function. Infusion of GnRH at a rate of 1 pulse/h restored menstrual cyclicity, with luteal phases of normal length and normal serum progesterone levels (5). Additional studies demonstrated that normal luteal phase length and progesterone production could be maintained with three pulses daily, but not with a single daily pulse during the luteal phase (6), delineating the lower limit of GnRH pulse frequency to maintain normal luteal function. However, few studies have addressed the amplitude of LH pulses required to maintain luteal function. Injection of LH or hCG several times daily during GnRH agonist or antagonist treatment to restore pulsatile gonadotropin activity has been performed by several investigators (2, 3), but maintenance of typical luteal phase progesterone levels through a luteal phase of normal length was not achieved. A reduction in the amount of GnRH infused into hypothalamus-lesioned monkeys at hourly intervals to decrease circulating LH also decreased serum progesterone and luteal phase length (7), demonstrating the dependence of proper luteal function and life span on the amount of LH per pulse as well as on the frequency.
Thus, studies examining natural (4) and experimentally controlled (6) menstrual cycles suggest that three LH pulses per day are appropriate and sufficient to maintain luteal function during the second half of the luteal phase, but the magnitude of LH pulses required to maintain luteal structure and achieve physiological levels of serum progesterone and relaxin, a protein hormone of the corpus luteum, throughout a luteal phase of normal length is not known. In the present study we administered the third generation GnRH antagonist antide to rhesus monkeys beginning on day 6 of the luteal phase to block endogenous LH release, as monitored by ablation of luteal progesterone production; then, various dose regimens of LH replacement were tested to determine the requirement for LH to restore normal luteal function and life span. Because a local role for LH-stimulated progesterone is suspected in the regulation of luteal structure and function (8, 9), and LH may regulate progesterone receptor (PR) expression in the primate ovary (10, 11, 12), luteal PR expression during LH ablation and replacement was also examined.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The general care and housing of rhesus monkeys (Macaca mulatta) at the Oregon Regional Primate Research Center (ORPRC) were described previously (13). Animal protocols and experiments were approved by the ORPRC animal care and use committee, and studies were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Adult females with regular menstrual cycles were checked daily for menstruation. Blood samples were obtained daily from unanesthetized monkeys by saphenous venipuncture from day 8 of the menstrual cycle. Serum was stored at -20 C. Day 1 of the luteal phase was defined as the first day of serum estradiol below 100 pg/mL after the midcycle estradiol peak (14). Daily serum progesterone levels and day of menstruation were also determined in 21 untreated regularly cycling monkeys from our colony (15).
To block pituitary LH release, the third generation GnRH antagonist antide [N-Ac-D-Nal1,D-pCl-Phe2,D-Pal3,Lys(Nic)5,D-Lys(Nic)6,Lys(iPr)8,D-Ala10] (16) was administered by sc injection in a vehicle of 50% propylene glycol and 50% water. Antide was synthesized at The Salk Institute and made available by the Contraceptive Development Branch, Center for Population Research, NICHHD. Based on published studies (17), monkeys were treated for 1 day with either 3 or 5 mg antide/kg BW or for 3 days with 3 mg antide/kg BW at 0800 h beginning on day 6 of the luteal phase. The latter treatment regimen (i.e. 3 mg/kg on days 6–8 of the luteal phase) successfully reduced serum progesterone to less than 1 ng/mL by day 8 and maintained low levels of serum progesterone throughout the remainder of the luteal phase. This dose and pattern of antide administration were used for all subsequent studies.
To titrate the amount of LH required to restore luteal function during and after antide treatment, recombinant human LH (Ares Advanced Technology, Randolph, MA) was dissolved in phosphate-buffered saline and injected at 0800, 1600, and 2400 h beginning on day 6 of the luteal phase and continuing until menstruation. Initially, animals received 5, 10, or 20 IU LH/injection. Based on these results, additional monkeys were treated with an escalating dose regimen of 5 IU LH/injection on days 6 and 7, 10 IU LH/injection on luteal days 8 and 9, and 20 IU LH/injection until menstruation.
To examine the effects of antide alone and with LH replacement on luteal structure and PR expression, additional monkeys were treated with antide alone, antide with a fixed dose of LH replacement (5 IU/injection), or antide with the escalating dose regimen of LH replacement (5 IU/injection on days 6 and 7 and 10 IU/injection on days 8 and 9). On day 10 of the luteal phase, corpora lutea were surgically removed (13). Portions of each tissue were placed in 10% formalin for histological examination, fresh-frozen in OCT (Tissue-Tek, Elkhart, IN) in liquid propane for immunocytochemistry, or flash-frozen in liquid N2 for preparation of ribonucleic acid (RNA).
Hormone assays
Serum levels of progesterone (18) and estradiol (19) were determined by RIA. Intra- and interassay coefficients of variation for the steroid RIAs did not exceed 15%. Serum relaxin levels were determined by homologous macaque relaxin enzyme-linked immunosorbent assay (20). Intra- and interassay coefficients of variation did not exceed 16%. LH levels were determined by mouse Leydig cell bioassay using monkey LH RP-1 (supplied by the NIH Hormone Distribution Program) as a standard (21). Intra- and interassay coefficients of variation for the LH bioassay did not exceed 19%.
Histology and immunocytochemistry
Formalin-fixed luteal tissues were embedded in plastic and sectioned for hematoxylin and eosin staining as previously described by the Morphology Core Laboratory at ORPRC (22). Immunocytochemical detection of PR in flash-frozen luteal tissues was performed after microwave fixation of tissue sections (23), using an antihuman PR antibody (JZB-39) (24). All luteal tissues were processed for immunocytochemical detection of PR in a single experiment.
PR messenger RNA (mRNA) analysis
Total RNA was isolated using the cesium chloride ultracentrifugation method of Chirgwin and colleagues (25). The ribonuclease protection assay to quantify PR mRNA was performed using 10 µg luteal total RNA and cyclophilin as an internal control, as previously described (26). Autoradiographs were scanned using a Hewett-Packard Scanjet 4c/T with Photoshop 4.0 and Twain software (Adobe Systems, Inc., Mountain View, CA) and were analyzed with Image 1.40 (NIH, Research Services Branch, NIMH, Bethesda, MD). Results from each experiment were normalized to the PR mRNA signal from monkey endometrial total RNA, which served as an interassay control.
Statistical analysis
Serum progesterone levels were log transformed before statistical analysis due to heterogeneity of variance, as determined by Bartlett’s test. Data were analyzed using two-way ANOVA with one repeated measure to compare the effects of both time and treatment. Unpaired t tests were used to compare serum progesterone levels in different groups on any single day. Because progesterone levels in monkeys receiving antide with the escalating dose regimen of LH replacement had a very small variance on day 11, these data were excluded from the ANOVA; comparison between these data and control progesterone levels on day 11 was made using the Mann-Whitney test. As immunoreactive relaxin concentrations vary widely between monkeys (27), serum levels during the treatment interval (day 7 to menstruation) were normalized to pretreatment levels (the average of days 1–6) for each individual animal. Relaxin concentrations were then expressed as a percentage of pretreatment levels. These data were log transformed and analyzed as described for progesterone. Serum LH levels were compared using one-way ANOVA with one repeated measure followed by Newman-Keuls test when indicated, using untransformed data. Luteal wet weights, PR mRNA content, and date of first menstruation were compared using one-way ANOVA followed by Newman-Keuls test when indicated, using untransformed data. All data were expressed as the mean ± SEM, and significance was assumed at P < 0.05.
Photography
Photomicrographs were made using Zeiss planapochromatic lenses (Carl Zeiss, New York, NY) and Ektachrome 64T film (Eastman Kodak Co., Rochester, NY).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), serum
progesterone fell below 1 ng/mL by day 7, but rebounded to
above 1 ng/mL on days 8–11, with menstruation occurring on day 14.
Because this dose failed to ablate luteal progesterone
production, additional animals received 5 mg/kg antide on day 6 of the
luteal phase (Fig. 1A). Again, serum progesterone levels
declined (1.5 ± 0.8 ng/mL) on day 7 and were below 1 ng/mL by day
8. In one of four monkeys treated with this dose of antide,
progesterone levels remained well below 1 ng/mL throughout
the remainder of the luteal phase, and menstruation occurred on day 9.
One of the other monkeys had serum progesterone levels of
approximately 1 ng/mL for several days after antide administration, and
the other two animals recovered normal progesterone
levels, with peak levels of 4.0 and 12.0 ng/mL on day 9 of the luteal
phase; these three monkeys menstruated on day 15.7 ± 0.7, similar
to untreated controls (day 16.3 ± 0.4). Because consistent
ablation of luteal function was not obtained with a single antide
injection at either dose, a series of three daily antide injections (3
mg/kg) was administered on days 6, 7, and 8 of the luteal phase (Fig. 1B). Serum progesterone declined to 1.2 ± 0.3 ng/mL
on day 7 (P < 0.05 compared to 6.5 ± 1.0 ng/mL
for controls) and remained below 1 ng/mL from day 8 through the end of
the luteal phase. These monkeys menstruated early on day 9.7 ±
0.3 (P < 0.05 vs. controls). This regimen
of antide administration was used in subsequent studies.
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), mean progesterone levels
were comparable to those measured in control monkeys for several days
after the initiation of treatment. However, serum
progesterone in monkeys receiving antide and 5 IU
LH/injection dropped below the 95% confidence interval for control
monkeys on day 10. By day 13, levels were markedly below control values
(0.4 ± 0.1 vs. 2.7 ± 0.6; P <
0.05), and the treated animals menstruated early (day 13.7 ± 0.9
vs. controls; P < 0.05). When LH was
administered at a dose of 10 IU/injection during and after antide
treatment (Fig. 2), serum progesterone levels were similar
to control values through day 12, then fell below the 95% confidence
interval for control monkeys on day 13. Monkeys receiving antide and 10
IU LH/injection also tended to menstruate early (day 14.3 ± 1.8)
compared to controls (P = 0.07). One monkey received
antide and 20 IU LH/injection (Fig. 2), and this treatment resulted in
progesterone levels above the 95% confidence interval for
control monkeys for 5 days after the initiation of treatment. Despite
continued LH administration, progesterone levels fell by
day 12, and this monkey also menstruated on day 14.
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). This pattern of LH replacement
increased progesterone levels above those measured in
animals receiving antide alone on days 7–13 (P <
0.05) and restored the normal pattern of serum
progesterone throughout the remainder of the luteal phase,
although progesterone levels were elevated above control
values on 1 day (day 11; P < 0.05). Monkeys treated
with the escalating dose regimen of LH menstruated on day 16 ± 1,
similar to control monkeys. Administration of antide alone also
resulted in very low serum relaxin levels (Fig. 3B), which did not
exhibit the expected rise during the second half of the luteal phase
(20, 27). In contrast, treatment with antide and the escalating dose
regimen of LH restored the rise in circulating relaxin previously
reported to begin on day 9 of the luteal phase in nonconceptive
menstrual cycles (27). Serum relaxin levels were elevated in antide
plus escalating LH-treated animals by day 8 (P < 0.05)
compared to monkeys treated with antide only; relaxin levels were
higher in antide plus escalating LH-treated animals throughout the
remainder of the luteal phase.
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). Thereafter, serum LH declined
by 8 h (P < 0.05) to levels not different from
those circulating before LH administration. In all cases,
administration of a single injection of 5 (not shown) or 10 IU LH
achieved serum LH levels in the expected range for the luteal phase in
naturally cycling rhesus monkeys (15), with peak LH levels generally
occurring at 1–2 h after and baseline levels achieved within 8 h
after LH administration.
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Additional studies examined the effects of antide alone and with
the suboptimal (fixed dose, 5 IU) and optimal (escalating dose) LH
replacement on primate luteal structure on day 10 of the luteal phase.
Treatment with antide alone for 3 days reduced luteal weight compared
to that in time-matched controls (P < 0.05; Table 1). Administration of antide with either
the fixed dose or the escalating dose regimen of LH replacement
resulted in luteal weights similar to control values.
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). On day 10 of
the luteal phase, luteal tissue from untreated monkeys had large luteal
cells, each with a round, centrally located nucleus and prominent
nucleolus (Fig. 5A). Some cells possessed vacuoles in the cytoplasm,
but large vacuoles were uncommon. Few nuclei were condensed or
crenated. Red blood cells were easily identified, indicating the
presence of capillary networks. In contrast, luteal tissue from
antide-treated animals contained very few large luteal cells (Fig. 5B),
and these cells possessed highly vacuolated cytoplasms. Remaining large
nuclei with prominent nucleoli were often surrounded by minimal
cytoplasm. Few nuclei were condensed or crenated, and red blood cells
were observed. Administration of either the fixed dose (Fig. 5C) or
escalating dose (Fig. 5D) regimen of LH replacement during and after
antide treatment maintained luteal structure similar to that seen in
time-matched control tissues; large luteal cells with intact nuclei and
red blood cells were frequently observed.
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; P < 0.05), while
treatment with the escalating dose regimen of LH during and after
antide administration maintained tissue PR mRNA at levels similar to
those seen in controls. Monkeys receiving antide and the fixed dose (5
IU LH/injection) of LH replacement had disparate luteal PR mRNA levels,
with the mean value intermediate between the antide alone and antide
plus escalating LH dose groups.
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). The use of the irrelevant antibody to
timothy grass pollen demonstrated that cytoplasmic staining was not
specific for PR (Fig. 7A, inset). On day 10 of the luteal
phase, many large luteal cells stained positive for PR, with brown
precipitate over the entire nucleus (Fig. 7A, arrow). After
treatment with antide alone (Fig. 7B), few PR-positive cells were
observed. However, some nuclei showed brown precipitate localized to
one portion of the nucleus (Fig. 7B, arrow). After treatment
with antide and the escalating dose regimen of LH (Fig. 7D) many large
luteal cells stained PR positive, similar to time-matched control
tissues. Staining was heterogeneous, however, as some portions of the
gland contained more PR-positive cells than others. Luteal tissue from
monkeys treated with antide and fixed dose LH replacement (Fig. 7C) had
some large luteal cells staining PR positive, but results were
intermediate between those obtained with antide treatment and the
control or treatment with antide and the escalating dose regimen of
LH.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Antide administration led to a loss of luteal function. In the present study, a single day of antide treatment transiently lowered serum progesterone levels, whereas treatment for 3 consecutive days successfully ablated luteal progesterone and relaxin production. Serum gonadotropin levels were not routinely measured, but previous studies by this laboratory (28) and others (17) indicate that antide prevents ovarian function by reducing circulating gonadotropin levels. When antide (0.5 mg/kg BW) was administered daily to macaques, LH levels were reduced within 2 days, and low LH and progesterone levels were maintained throughout the treatment interval (28). Although Gordon and colleagues (17) showed that a single administration of antide (3 mg/kg BW) to cynomolgus monkeys at the midluteal phase rapidly lowered serum LH below the level of detection for 6 days and maintained low serum progesterone for the remainder of a luteal phase of normal length, our data indicate that sustained treatment with antide at this dose is required to ablate progesterone production by rhesus macaques. The present (data not shown) as well as other (17) studies indicate that monkeys resume menstrual cyclicity within a few months after cessation of antide treatment, suggesting that administration of antide is an effective technique to rapidly and reversibly ablate LH.
Antide treatment decreased the wet weight of the corpus luteum and luteal cell size, indicating that luteal structure as well as function require continued gonadotropin support. Structural luteolysis near the end of the spontaneous menstrual cycle also includes decreased luteal weight and cell size (29, 30) and correlates with the cessation of luteal function. Although spontaneous luteolysis and GnRH antagonist exposure elicit some common changes in luteal structure in macaques, a notable exception was the observation of nuclear breakdown during spontaneous luteolysis (30), but not during antide treatment (current study). However, GnRH antagonist administration to marmosets 2 days before the removal of the corpus luteum elicited luteolytic and necrotic changes, including nuclear breakdown (31), so the timing of the observation may be critical for the detection of nuclear and other changes in response to gonadotropin deprivation. Spontaneous luteolysis occurs despite continued gonadotropin stimulation, but whether premature gonadotropin withdrawal initiates processes within the corpus luteum similar to those that occur during spontaneous luteolysis is unknown. In addition, gonadotropins can restore luteal function after both gonadotropin deprivation at the midluteal phase (3, 32) and spontaneous luteolysis (33), further complicating the definition of structural vs. functional luteolysis and the role of gonadotropin in determining luteal life span.
Few studies have addressed the critical features of LH pulse frequency and amplitude during the luteal phase in primates. Studies using hypothalamus-lesioned, GnRH-replaced monkeys demonstrated that GnRH (and, presumably, LH) pulses during the luteal phase at frequencies ranging from hourly to every 8 h were able to maintain luteal progesterone production through a luteal phase of normal length in most monkeys, but a single pulse every 24 h was inadequate to maintain sufficient progesterone production for overt menstruation to occur upon withdrawal (6). Infusion or injection of LH has been used to examine the role of LH in the maintenance of the primate corpus luteum, with LH administration every 3 (2) or 8 (34) h sufficient to stimulate luteal progesterone production. In these experiments, LH pulse amplitude (i.e. the amount of LH per administration) was held constant. In one study using hypothalamus-lesioned monkeys, LH pulse amplitude was modulated by controlling the amount of GnRH infused with each pulse. Hourly GnRH pulses of sufficient amplitude maintained menstrual cyclicity, but suboptimal amounts of GnRH per hourly pulse led to decreased LH and progesterone levels (7); more than half of these animals menstruated early. However, the amplitude of LH pulses was not measured and may have varied across the luteal phase. In the present study, constant pulse amplitude achieved with fixed dose LH administration during the second half of the luteal phase did not maintain luteal progesterone production through a luteal phase of normal length regardless of the amount of progesterone production stimulated. In contrast, the escalating dose regimen of LH administration maintained serum progesterone levels through a luteal phase of normal length. Taken together, these studies suggest that when pulse frequency is above a minimal threshold, LH pulses of increasing amplitude may be required for continued progesterone production through a luteal life span of normal length.
LH administration yielded serum LH levels with the characteristics of normal luteal LH pulses. Peak LH levels occurred within 1–2 h after administration, and serum LH returned to pretreatment levels within 8 h, with peak LH levels similar to those measured during the luteal phase of natural menstrual cycles (15). Previous studies have demonstrated that the serum clearance and half-life of recombinant human LH and LH-like gonadotropins are dose independent (35, 36), so it is unlikely that LH persisted in the serum after 8 h at any dose used in the present study. Protocols involving the administration of higher doses of recombinant human gonadotropins (FSH, LH, and hCG) to stimulate follicular development did not cause the generation of antigonadotropin antibodies after 7–9 days of exposure until at least the second protocol (our unpublished data), suggesting that the declining progesterone levels measured in the present study after days of low dose LH administration were not due to inactivation of gonadotropin by antibodies.
Increased LH pulse amplitude during the later stages of the luteal phase may be required to stimulate progesterone production and other functions by aging luteal cells. Early observations (37) that progesterone production by cultured luteal cells in response to a maximal dose of hCG declined over the course of the luteal phase led to further experiments examining gonadotropin-stimulated cAMP production by the corpus luteum. Luteal LH receptor concentration (38) and basal as well as gonadotropin-stimulated adenylyl cyclase activity (39) were maximal on days 6–12 of the luteal phase, with no change in LH receptor affinity for gonadotropin during this interval (38). Therefore, the increased requirement for LH during this period in vivo is not likely to be due to decreased activity of the gonadotropin receptor-adenylyl cyclase signaling system. However, by luteal days 13–15, both LH receptor concentration (38) and hCG-stimulated adenylyl cyclase activity (39) were decreased compared to midluteal levels, suggesting a possible mechanism for the decreased gonadotropin responsiveness of aging luteal cells. The increasing amplitude of LH pulses during the second half of the luteal phase (4) may be responsible for maintaining luteal progesterone production during the second half of the luteal phase. However, decreasing luteal cell sensitivity to LH may still result in luteolysis, as luteal regression proceeds despite increasing LH pulse amplitude during the late luteal phase.
The present study supports a role for LH in the maintenance of normal PR expression in the corpus luteum of the menstrual cycle. GnRH antagonist treatment to ablate LH increased PR mRNA but decreased PR immunostaining, whereas LH replacement restored PR mRNA and protein to control levels. Previous studies indicated that the ovulatory LH surge promoted PR expression in the luteinizing granulosa cells of the follicle in monkeys (24, 40), women (41), and rodents (42); in vitro exposure to gonadotropin also stimulated PR expression in luteinizing granulosa cells from a variety of species (10, 11, 12). These data suggest that gonadotropin is a major regulator of PR expression in the developed corpus luteum as well as in the luteinizing follicle. Luteal PR mRNA increased while PR protein levels decreased during the natural luteal phase (26, 43) and in simulated early pregnancy (29) as well as in response to gonadotropin deprivation (current study), suggesting that critical regulation of luteal PR content is not at the level of mRNA but is, instead, posttranscriptional. In addition, progesterone deprivation during LH stimulation modulates primate luteal PR expression (8, 26), highlighting the importance of differentiating between the direct effects of LH and those mediated by LH-stimulated steroids in the corpus luteum.
In summary, we have developed a model of GnRH antagonist administration with an escalating dose regimen of LH replacement that maintains the structure as well as the steroidogenic and peptidergic functions of the primate corpus luteum through a luteal phase of normal length. This nonsurgical, reversible method of LH ablation and replacement will allow further studies examining the role of LH in the regulation of specific structural and functional properties of the primate corpus luteum. Studies supporting a role for progesterone in the regulation of luteal structure (8), progesterone production (8, 44), and LH receptor expression (9) could be advanced by using this model in conjunction with steroid synthesis inhibitors (8) to further elucidate the role of progesterone in the regulation of the structure and functional life span of the corpus luteum in an environment of sustained gonadotropin support.
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Acknowledgments |
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Footnotes |
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Received May 6, 1998.
Revised September 23, 1998.
Accepted September 30, 1998.
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