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a Departments of Obstetrics/Gynecology and Neurobiology, and the Laboratory of Neuroendocrinology of the Brain Research Institute, University of California, Los Angeles School of Medicine, Los Angeles, California 90095
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In contrast to the cyclic increases in P4, estradiol (E2), LH, and FSH in control animals, P4-implanted rats exhibited elevated serum P4 but low E2, LH, and FSH levels. After implant treatments, the follicular reserve was significantly (p < 0.05) larger in P4-treated females (2012 ± 297 resting follicles per ovary, n = 5 rats per group) than in regularly cyclic control rats (713 ± 226 follicles per ovary, n = 7). The effects of P4 implants on the follicular reserve were associated with a subsequently higher incidence of regular estrous cycles after P4 treatment. These results demonstrate that P4 prevents cyclic increases in E2 secretion and is associated with a conservation of the ovarian follicular reserve and the maintenance of regular estrous cycle patterns, indicating a protective effect of P4 on the age-related loss of ovarian follicles.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous studies indicate that the progression of reproductive aging may be modulated by the pattern of exposure to ovarian steroids. The cessation of regular estrous cycles occurs significantly earlier in life in virgin rats than in multiparous females [3, 11]. In addition, our previous studies have shown that successive treatments of young virgin rats with progesterone (P4) implants decrease estradiol (E2) secretion and result in the extended maintenance of regular estrous cycles and fertility [11]. Ovariectomy of rats at a young age prevents the age-related loss of neuroendocrine function [12]. These results may suggest that the repeated, large increases in E2 secretion during successive estrous cycles eventually render the hypothalamic-pituitary axis unresponsive to further positive feedback action of E2 on LH secretion. Thus, repeated treatments with P4 implants may act to preserve a normal positive feedback response by reducing circulating E2 levels [11]. In consonance with this view, concomitant treatments with E2 implants significantly counteract the beneficial effects of P4 implants on reproductive aging [13].
In addition to the pattern of steroid exposure, the size of the ovarian follicular reserve has been shown to influence the progression of reproductive aging. Experimental reduction of the follicular reserve by unilateral ovariectomy advances the loss of regular cyclicity and fertility in rodent species [14, 15]. In addition, food restriction delays the onset of reproductive aging and is associated with a delayed loss of primordial follicles from the ovaries [16, 17]. While the mechanisms regulating the rate of follicular recruitment and depletion are not understood, it is possible that the influences of repeated P4 implant treatments on reproductive aging are associated with changes in the rate of follicular loss. To examine such a possibility, the present study was undertaken to reveal the effects of successive P4 implant treatments on age-related changes in the ovarian follicular reserve and subsequent estrous cyclicity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Long-Evans virgin female rats (85 days old) were obtained from Charles River Laboratories (Portage, MI) and housed five per cage under standard vivarium conditions, with room temperature (24–26°C) and lighting (lights-on from 0500 to 1900 h daily) controlled throughout the study. Daily vaginal smears were taken from these rats to determine their estrous cycle patterns. Animals that exhibited at least three consecutive 4- to 5-day-long estrous cycles were considered to be regularly cyclic and were used for the following experiments. Experiments were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction.
P4 Implant Treatments
Beginning at 100 days and continuing until 8 mo of age, female rats received s.c. Silastic (Dow Corning, Midland, MI) implants containing P4 (P4-implanted; n = 55) or empty capsules (control; n = 59). Implants were in place for 3 wk; this was followed by removal of implants for 1 wk. The P4 implants were prepared by packing Silastic tubing (4 cm in length, 0.333 cm i.d.) with 100% crystalline P4 (Sigma Chemical Co., St. Louis, MO) to attain serum P4 levels of 30–50 ng/ml [11, 13]. Placement and removal of these implants were performed while the rats were under acute, light ether anesthesia. Implant treatments were repeated five times in these same groups of animals until 8 mo of age.
Characterization of Circulating Hormone Levels during Implant Treatments
The daily patterns of circulating hormones were examined in separate groups of 4-mo-old virgin rats (n = 6–10 per group) treated with P4 implants or empty implants. Consecutive blood samples (1.0 ml each) were obtained from these rats at 1000 h prior to implant placement, at 1800 h of that day, and at 1200 h on the next 2 days. Thereafter, samples were taken at 1200 h once every 2 days until the end of implant treatment. Samples were obtained by cardiac puncture under acute, light ether anesthesia. Blood was kept at 4°C overnight and then centrifuged, and serum was separated and stored at -20°C until assayed for E2, P4, LH, and FSH values by RIAs. For comparison, samples were also obtained from cyclic control females (4–5 mo of age) at 1200 h of each day of the estrous cycle. To further reveal the patterns of gonadotropin secretion during steroid implant treatments, additional experiments were performed on separate groups of control and P4-implanted rats. Young virgin rats (n = 8 rats per group) received P4 implants as above, and 9 days after implant placement a jugular vein catheter was surgically placed as previously described [18]. Two days later, sequential blood samples (0.4 ml each) were taken from these females through the catheter at 0800, 1100, 1400, 1700, and 2000 h. Serial blood samples were also taken from cyclic female controls on diestrous Day 1 and on proestrus (n = 5 per group). Blood was collected into a heparinized syringe and centrifuged immediately. Plasma was separated and stored at -20°C until assayed for LH and FSH.
Influence of P4 Implant Treatments on the Ovarian Follicular Reserve
During the last implant treatment (at 8 mo of age), ovaries were collected from some female rats in each group to determine the influence of steroid implant treatments on the age-related loss of ovarian follicles. Ovaries were taken from P4-implanted rats (n = 5 per group) 20 days after administration of the last implants, and from cyclic virgin controls (n = 7) at 1200 h on diestrous Day 1. Ovaries were fixed in Bouin's solution and processed to obtain paraffin sections of 8-µm thickness. Sections were stained with hematoxylin and eosin, and every sixth section was examined at x400 magnification to count the numbers of resting (primordial and small primary) follicles (< 50 µm in diameter). The total number of resting follicles was determined by multiplying the sum of the counted follicles by six, a method similar to that previously described [17]. We have found that the total number of resting follicles determined by this method is comparable to that found through examination of serial sections of the same ovary (data not shown). All slides were coded and randomized so that treatment groups of each rat were unknown at time of analysis.
Influence of P4 Implant Treatments on the Subsequent Incidence of Regular Estrous Cyclicity
After removal of the last implants at 8 mo of age, subsequent patterns of estrous cyclicity were determined by daily vaginal lavage for the next 4 wk. Rats that displayed prolonged cycles (6 or more days) of variable duration were considered to be irregularly cyclic, whereas those displaying more than 15 consecutive days of vaginal cornification were classified as persistent-estrous.
Hormone Assays
Plasma concentrations of LH and FSH were measured by double-antibody RIAs, as previously described [19], employing reagents provided by the National Hormone and Pituitary Program, NIDDK (Baltimore, MD). LH and FSH values are expressed in terms of the reference standards NIDDK rat LH-RP-1 and rat FSH-RP-1, respectively. The intra- and interassay coefficients of variation were 3.2% and 7.9%, respectively, for LH and 8.9% and 10.9%, respectively, for FSH. Serum concentrations of E2 and P4 were measured by specific RIAs as previously reported [19]. Prior to RIA, samples were extracted with diethyl ether, and P4 and E2 fractions were isolated by Celite (Celite Corporation, Lompoc, CA) column chromatography [20]. The intra- and interassay coefficients of variation were 5.4% and 6.9%, respectively, for E2 and 5.7% and 7.2%, respectively, for P4.
Statistical Analyses
Comparisons of the numbers of resting follicles among the treatment groups were performed by one-way ANOVA, followed by Duncan's multiple range test when appropriate. Two-way ANOVA was employed to compare the plasma patterns of LH and FSH during 0800–2000 h of the day in female rats implanted with P4 and those with empty implants. The incidences of regular cyclicity in each group were compared by chi-square test. A confidence level of p < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Figure 1 depicts the daily patterns of serum E2 in rats during 3 wk of treatments with P4 implants (left panel), compared to those in cyclic females on different days of the estrous cycle (1200 h; right panel). Cyclic females exhibited elevated serum E2 (86 ± 6 pg/ml) at noon on proestrus. In young rats, s.c. placement of a P4 implant resulted in a rapid and consistent decrease in serum E2, ranging between 19 ± 1 and 27 ± 7 pg/ml, similar to that previously reported [11, 13]. These low E2 values in P4-implanted rats were comparable to the levels seen on estrus and diestrous Day 1 in cyclic females, but were substantially lower than proestrous E2 concentrations. Thus, placement of P4 implants prevented the cyclic increases in E2 observed in control females.
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Figure 1 also compares the patterns of serum P4 between P4-implanted females and cyclic control rats. During the estrous cycle, serum P4 levels fluctuated at noon between 25 ± 3 and 40 ± 4 ng/ml. Placement of P4 implants in female rats produced a rapid and marked rise in serum P4 (139 ± 13 ng/ml) within 8 h, followed by a gradual decline that reached basal levels by Day 12. These data demonstrate that circulating concentrations of P4 were significantly elevated during the first half of P4 implant treatments, as compared to the levels observed at noon during the estrous cycle.
Figure 2 depicts the daily patterns of serum LH levels in rats implanted with P4 for 3 wk as compared to regularly cyclic controls. During the estrous cycle, serum LH values at noon fluctuated between 103 ± 11 and 125 ± 5 ng/ml. Similarly low levels of LH were observed in P4-implanted rats. To examine in more detail the patterns of LH secretion in these groups, serial samples were obtained every 3 h between 0800 and 2000 h (Fig. 3). While cyclic controls displayed a distinct preovulatory LH surge on proestrus, P4-implanted rats showed consistently low plasma LH levels similar to those seen in cyclic controls on diestrous Day 1. Figure 2 also depicts the daily patterns of serum FSH in both groups of animals. During the estrous cycle, serum FSH values at noon were significantly (p < 0.05) higher on estrus than at other stages, whereas P4-implanted females exhibited low serum FSH values throughout the 3-wk period. Figure 3 (bottom panel) further reveals the plasma patterns of FSH during a 12-h period in these three groups of rats. While cyclic controls exhibited a distinct preovulatory FSH surge on proestrus, P4-implanted rats showed relatively stable low levels of plasma FSH, similar to that observed on diestrous Day 1. Thus, P4-implanted females did not exhibit cyclic increases in gonadotropin secretion during hormone treatment, consistent with the persistently low levels of E2 and elevated P4 observed in these animals.
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Effects of Successive P4 Implant Treatments on Subsequent Patterns of Estrous Cyclicity during Aging
Figure 4 depicts the incidence of regular estrous cyclicity in female rats treated with P4 implants, as compared with virgin controls. At 9 mo of age, only 21% of control females were regularly cyclic, the remainder displaying irregular cycles (14%) or persistent estrus (65%; data not shown). In contrast, rats previously treated for over 4 mo with successive P4 implants subsequently exhibited a significantly greater incidence of regular cyclicity (58%) at 9 mo of age than controls (p < 0.05). The maintenance of regular cyclicity among P4-treated rats was reflected by a decreased incidence of persistent estrus (26%; data not shown) compared to that observed in controls (65%).
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Influences of P4 Implant Treatments on the Ovarian Follicular Reserve
To examine possible influences of steroid implant treatments on the age-related loss of ovarian follicles, histological analyses were performed on ovaries obtained during the last implant treatment (8 mo of age). Ovaries from cyclic controls exhibited follicles at various stages of development and showed prominent corpora lutea (data not shown). In contrast, although P4-implanted females also displayed numerous nonatretic follicles in early stages of development (up to 250 µm in diameter), no corpora lutea were evident, consistent with the absence of ovulation during P4 treatment. As shown in Figure 5, ovaries from 8-mo-old cyclic control females had 713 ± 226 resting (primordial and small primary) follicles (< 50 µm in diameter) per ovary (n = 7). In contrast, P4-implanted rats had 2012 ± 297 resting follicles per ovary (n = 5), significantly more than in controls (p < 0.05). Thus, the effects of successive P4 implants on the subsequent maintenance of regular cyclicity were associated with the absence of corpora lutea formation and delayed loss of follicles from the ovarian follicular reserve during P4 implant treatment.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the female rat, as in the human, there is an age-related decline in the ovarian follicular reserve [10, 23, 24]. Several studies indicate that experimental reduction of the ovarian follicular reserve, such as that achieved by unilateral ovariectomy, accelerates the onset of reproductive aging in the rat [14, 15]. Similarly, the loss of regular cyclicity in middle-aged mice appears to be related to diminished numbers of resting follicles in the ovary [24–26]. Middle-aged cyclic rats display reduced numbers of developing follicles during the estrous cycle [22], although the mean diameter and estradiol content of these developing follicles are greater than in young animals [22]. Thus, an age-related decline in the ovarian follicular pool is associated with alterations in the pattern of follicular development and steroidogenesis, with potential impacts upon neuroendocrine function and oocyte quality. In light of these previous observations, it is conceivable that there is a functional correlation between the dramatic effects of P4 implant treatments on the maintenance of regular estrous cyclicity and on the conservation of the ovarian follicular reserve during aging.
Little is known regarding the factors that may regulate the rate at which primordial follicles are recruited from the resting pool for development. It has been reported that hypophysectomy delays the rate at which ovarian follicles are lost during aging, suggesting a role of gonadotropins or other hypophysial factors in regulating follicular recruitment [27, 28]. In the present study, chronically elevated circulating P4 levels in P4-treated rats prevented cyclic increases in both FSH and LH secretion, although basal levels of gonadotropins were similar to that seen during the estrous cycle. In addition, elevated circulating P4 levels were associated with the absence of cyclic rises in ovarian E2, presumably resulting from inhibitory effects of P4 on FSH-induced aromatase activity [29] or prevention of cyclic increases in FSH release during implant treatment. Under this hormonal environment, follicular recruitment was apparently decreased, such that at 8 mo of age the P4-implanted rats had a follicular reserve three times greater than that of cyclic control females. Further studies are required to determine whether the marked effects of P4-implant treatments on follicular recruitment are directly due to the effects of elevated P4, the absence of cyclic increases in E2 and gonadotropins, or some combination thereof.
In women, there has been little evidence to indicate a sparing effect of oral contraceptive use on the age of menopause. One recent report [30] indicates that women with a history of oral contraceptive use have lower cycle Day 3 FSH values than women with no prior history. Since this FSH value is believed to be predictive of the ovarian follicular reserve [31–33], these findings may suggest some influence of oral contraceptives on the rate of follicular recruitment. In this regard, it is important to note that we began P4 implant treatments relatively early and implemented them over a relatively long period in the reproductive life span of the female rat. Thus, variations in the age at which oral contraceptive use is initiated, and the consistency of such use, may make it difficult to determine significant effects of steroids on the rate of follicular loss in women. In addition, most oral contraceptives contain low amounts of estrogens. In this regard, we have demonstrated that in female rats, cotreatment with E2 counteracts the protective effects of P4 implants on reproductive aging [13]. Thus, the low amounts of estrogens in oral contraceptives may counteract potential protective effects of P4 on follicular loss. It is also not clear whether the progesterone derivatives used in contraceptives would have the same protective effects as P4 used in this study. Nevertheless, future studies of women who receive long-term treatments with progestogen implants (Norplant) for contraceptive purposes may provide more insight into the role of elevated P4 on follicular recruitment and the onset of menopause.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: John K.H. Lu, Department of Obstetrics & Gynecology, Rm. 22–177 CHS, UCLA School of Medicine, Los Angeles, CA 90095–1740. FAX: (310) 206–6531.
3 Current address: Department of Biology & Microbiology, California State University Los Angeles, Los Angeles, CA 90032.
4 Current address: Department of Obstetrics/Gynecology, Medical College of Virginia, Richmond, VA 23298.
Accepted: March 9, 1998.
Received: December 19, 1997.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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