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Neuroendocrinology |
Departments of Obstetrics/Gynecology5
Neurobiology6 and
the Laboratory of Neuroendocrinology of the Brain Research Institute,7 D. Geffen School of Medicine at University of California, Los Angeles, California 90095
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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aging, follicle-stimulating hormone, luteinizing hormone, neuroendocrinology, progesterone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Beginning at 9–10 mo of age, increasing numbers of multiparous female rats display significantly attenuated preovulatory LH surges before any change in the regular 4-day estrous cycle [8–11]. Within weeks, females that exhibit such attenuated LH surges lose their regular cyclicity, as revealed by a lengthened, irregular estrous cycle with an extended estrous phase [11, 12]. Thus, the age-associated change in estrous cyclicity is accompanied by a delay in both LH surge and ovulation, and it may be caused by a decreased hypothalamic response to the positive-feedback action of E2 [13]. Soon thereafter, all middle-aged, irregularly cyclic female rats enter an acyclic, persistent-estrous (PE) state, in which the animal lacks both spontaneous ovulation and the preovulatory LH surge [12, 14, 15], that may last for 6 mo or longer. During this acyclic PE state, the animal is chronically exposed to somewhat elevated circulating E2 and diminished P4, except for the small contribution of P4 by the adrenal [15], and such elevated E2 leads to the persistent presence of cornified epithelial cells in the vaginal smear profile. At present, it remains unknown whether the absence of an LH surge in middle-aged PE females results from a defect in the hypothalamic-pituitary responsiveness or involves changes in the hormonal milieu. Notably, the proestrous rise in P4 is consistently reduced in middle-aged animals that have regular cycles and display an attenuated LH surge [9], and P4 production is even more markedly diminished at cycle cessation [16]. Thus, P4 deficiency in middle-aged animals may be an important hallmark of reproductive aging.
In early PE animals, the gonadotropin surge response to the stimulation of E2 alone is no longer effective [17, 18]. These animals remain sexually receptive to male rats [19, 20], however, and mating in a subset of PE animals will induce a more prompt increase in plasma P4, followed by ovulation [18]. These results establish a temporal relationship between the LH surge and P4 secretion during PE, and they support a role for P4 in regulation of the LH surge. Thus, the neuroendocrine mechanism that elicits the LH surge may still be intact during early PE, but a concomitant increase in P4 level may be required to control the mating-elicited LH surge [18, 19]. This contention also agrees with the results of previous studies regarding the inability to elicit preovulatory gonadotropin surges in response to E2 stimulation during the PE-associated loss of estrous cyclicity. Whereas long-term ovariectomy (OVX) restores the LH surge-inducing action of E2 with P4 [21, 22], this ability disappears again soon after chronic E2 implantation [21]. Thus, the neuroendocrine dysfunction during PE may relate to chronic E2 exposure in the absence of a concomitant rise in plasma P4 rather than to aging per se. This observation is supported by evidence that chronic exposure of young rats to E2 mimics loss of the LH surge-inducing actions of E2, either alone or with P4, similar to PE [22]. Moreover, the responsiveness to E2 alone and of E2 plus P4 disappears after 2 and 4 wk, respectively, of chronic E2 administration [22].
Collectively, these observations prompted the current examination of the PE state, in which we hypothesized that middle-aged PE rats lose their ability first to respond to E2 soon after PE onset and then to respond to E2 plus P4 during the extended PE duration. To examine our hypothesis, we first assessed whether a difference exists between early and long-term PE stages regarding the gonadotropin surge responses to E2 stimulation and P4 challenge in female rats that had entered a PE state within 35 days, and we compared these responses to those of long-term (>80 days) PE animals. We also examined the role of P4 in the gonadotropin surge response to mating with a fertile male.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Multiparous female rats (
8–9 mo of age as retired breeders, n = 60) of the Long-Evans strain were purchased from Charles River Laboratories (Portage, MI) and maintained in our animal facility under controlled temperature (24–26°C) and lighting (14L:10D; lights-on, 0500–1900 h) conditions. In the vivarium, rats were grouped five per cage, and Purina chow (Purina Mills, St. Louis, MO) and drinking water were provided ad libitum. For the caging/mating studies, 15 young, fertile male rats were also maintained in separate cages under the same vivarium conditions. All animal 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.
The vaginal cytology of multiparous rats was evaluated for more than 2 mo (68 ± 6 days, mean ± SEM) to determine their estrous cycle patterns. Females that had become irregularly cyclic were selected and followed until they exhibited PE for at least 15, but no longer than 35, consecutive days (i.e., early PE). After monitoring, 11- to 12-mo-old females had gone through the transition from regular to irregular cyclicity. From a colony of these animals, we identified several subsets of multiparous female rats for our studies (Table 1).
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Early PE rats (n = 13) Early PE rats were selected as described, with a mean PE duration of 26.5 ± 1.6 days. Among these 13 early PE animals, 7 were challenged with P4 (s.c. injection) under E2-primed conditions (EP group), and the remaining 6 received corn oil vehicle as controls (EO group).
Long-term PE rats (n = 6) The remaining animals in the colony were kept in the vivarium until 15–16 mo of age, and the daily vaginal smears on each female rat were continuously monitored. Within this group, six female rats were identified as long-term PE (LP group), because each of these females had exhibited 70–96 consecutive days of estrous vaginal smears (with a mean PE duration of 81.5 ± 10.2 days, n = 6). Certain considerations limit the availability of such long-term PE rats with a complete estrous cycle record. Specifically, the fragile health of rats after the age of 12 mo leads to substantial morbidity/mortality during the jugular vein cannulation procedure. Moreover, the costs of long-term housing and daily estrous cycle monitoring made a large study of this cohort prohibitively expensive. Therefore, all six LP females were used exclusively for testing with a P4 challenge.
Jugular Vein Cannulation
Following subset identification, animals underwent bilateral OVX under light ether anesthesia and were given a jugular vein catheter. Immediately following cannulation, a plasma sample was taken for E2 and P4 measurement, and then each animal received an s.c. E2 implant, as described below.
To characterize the plasma patterns of gonadotropin surges after E2 and P4, it was necessary to take multiple blood samples from the tested animals for both LH and FSH RIAs. Therefore, after identification of PE animals, each female was surgically implanted with a jugular vein catheter under light ether anesthesia (Day 0) using a slight modification of previously described methods [23, 24]. Each catheter was made from two different sizes of Medical Grade tubing (SF Medical, Hudson, MA); a 6-cm length of smaller tubing (inner diameter, 0.025 inch) was jointed to a 10-cm length of larger tubing (inner diameter, 0.03 inch). The junction of these two pieces of tubing was glued and secured by Silastic Medical Adhesive (silicone type A; Dow Corning, Midland, MI). The intravascular portion (i.e., smaller-sized end) of the catheter was trimmed and inserted into the right jugular vein. Then, the catheter was gently pushed up to the junction and anchored in place with several sutures. The opposite end (i.e., larger-sized end) of the catheter was passed s.c. to exit the skin just between the ears. The cannula tubing was filled with heparinized sterile saline. The end of this exit tubing was closed by insertion of a 1-cm plug made from the hub of a 25-gauge needle. If a catheter is properly placed in the jugular vein and secured, it can remain patent for 10–15 days. This procedure allows the collection of sequential blood samples over several days while the animal is both conscious and freely moving. Moreover, to prevent anemia, blood cells are separated from the plasma needed for hormone analysis and then infused back into the same animal.
To ensure patent cannulas in all animals tested throughout the 4-day duration of the experiments, blood collection immediately following cannulation on Day 0 was necessary. Thus, shortly before OVX at 1200 h on Day 0 and again at noon on Days 3 and 4, a 0.5-ml blood sample was taken from each animal through an implanted jugular vein catheter (see above). This procedure required blood samples to be collected while the animals were under surgical stress, which may be reflected in the unusually high levels of P4 on Day 0 (see Fig. 1B). The blood was centrifuged, and the plasma was saved and stored at –20°C until assayed for LH, FSH, E2, and P4 by RIA.
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Silastic E2 Implants
The E2 implants were prepared by packing a piece of silastic medical tubing (length, 3 mm; inner diameter, 1.57 mm; Dow Corning) with a mixture of crystalline E2 and cholesterol (50% E2:50% cholesterol by weight; Sigma Chemical Co., St. Louis, MO). Before use, silastic E2 implants were incubated in physiological saline (0.9% NaCl) at 37°C overnight both to remove excessive E2 on the outside surface of the implant, thereby minimizing a surge release of E2 after implant placement, and to check the seal [21, 25].
P4 Treatment
On Day 4, immediately after blood sampling at 1200 h, these rats received an s.c. injection of either P4 (EP group: 0.5 mg/100 g body weight [bw], n = 7) or vehicle (EO group: 0.1 ml corn oil/100 g bw, n = 6). Beginning at 1400 h on that afternoon, serial blood samples (0.3 ml each) from each rat were taken once every 30 min during 1400–1600 h and then hourly during 1600–2000 h and every 90 min during 2000–2300 h. Blood was collected into a 1-ml tuberculin syringe coated with heparin and then centrifuged as described above. Plasma was separated and stored for subsequent P4, LH, and FSH assays. For long-term PE females (LP group: n = 6), each animal was injected with P4 at 1200 h and sampled using methods similar to those used with the early PE rats.
Caging of Early PE Females with Fertile Males
In addition to the experiments described above, separate experiments were also performed to examine the ability of caging/mating with males to produce gonadotropin surges in PE females that received P4 versus oil treatment, because mating can release gonadotropins in the female rat [26]. Mating frequently results in disruption of the PE pattern of vaginal smears or in pseudopregnancy [14, 27, 28]. A similar cohort of 15 early PE rats was recruited and received the same surgical protocol as those in the EP and EO groups described above (Table 2). At 1200 h on Day 4, eight females were each injected with P4 (EPM group: 0.5 mg/100 g bw, s.c.), whereas the other seven animals received the vehicle corn oil (EOM group: 0.1 ml/100 g bw, s.c.) as a control (Table 2).
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Beginning at 1330 h, a young, fertile male was placed into a transparent Plexiglas viewing box (length, 50.5 cm; width, 50.5 cm; height, 40.5 cm) to allow animal acclimatization to the box. At 1400 h, a blood sample (0.3 ml) was taken from all PE female rats via an implanted jugular vein catheter. Immediately after sampling, both EOM and EPM rats were individually introduced to the box and caged with a fertile male rat until 1900 h. When caged with males, the lordotic behavior of each female rat was observed and recorded as previously described [17], including the time that the female took to show the first lordosis, the number of lordosis postures by the female, and the number of mounts by the male (Table 2). These values were then used to establish the lordosis quotient, which was defined as the number of lordosis postures by the female divided by the number of mounts by the male [17, 18]. During that afternoon, from 1400 h until 2330 h, female rats were removed from the box for serial sampling at the previously described time points.
Hormone Assays and Statistical Analyses
Plasma concentrations of both LH and FSH were measured by double-antibody RIA as previously described [16, 21] using reagents provided by the National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK, Baltimore, MD). Plasma levels of LH and FSH were expressed in terms of the reference standards NIDDK rat LH-RP-2 and FSH-RP-1, respectively. The intra- and interassay coefficients of variation were 3.2% and 7.6%, respectively, for LH and 8.2% and 10.3%, respectively, for FSH. Plasma levels of E2 and P4 were measured by specific RIA methods as previously described [15, 29, 30]. Before the assays, each plasma sample was extracted with diethyl ether, and the P4 and E2 fractions were separated from other steroids using Celite (Celite Corporation, Lompoc, CA) column chromatography as described by Brenner et al. [30]. The intra- and interassay coefficients of variation were 5.1% and 6.5%, respectively, for E2 and 5.4% and 7.0%, respectively, for P4. Statistical analyses of possible differences in animal body weight and in the duration of PE were performed by one-way analysis of covariance (ANCOVA). Statistical differences in plasma LH, FSH, E2, and P4 levels over time and among the EO, EP, and LP groups were assessed by two-way ANOVA with repeated measures followed by the Tukey test for post-hoc comparison. A confidence level of P < 0.05 was considered to be statistically significant. For the male caging/mating studies, statistical differences in plasma LH, FSH, E2, and P4 levels over time and between the EOM and EPM groups were similarly assessed. The low number of LP animals necessitated the assessment of fewer time points, so to compare these animals with the EP group (see Fig. 4), the values of the EP time points were averaged to match the sampling schedule of the LP animals.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The data in Tables 1 and 2 show that both the body weight and the duration of PE remained remarkably similar among the four subgroups of early PE rats (EO, EP, EOM, and EPM groups). In contrast, the long-term PE females (LP group) had higher body weight (P < 0.05) than the early PE animals at the time of the experiment and blood sampling.
Elevated Plasma E2 Level in PE Rats before and after OVX and E2 Implantation
Figure 1 shows the plasma patterns of E2 before and after OVX and E2 implantation in PE animals (EO, EP, and LP groups). Before OVX (Day 0), the mean concentrations of plasma E2 ranged between 35 and 46 pg/ml for the three groups. Subsequent placement of the E2 implant in OVX animals produced an initially elevated plasma level of E2 on Day 3 (53–62 pg/ml) that was significantly higher than the level on Day 0 (P < 0.01) before OVX. However, by Day 4, circulating E2 concentrations were in a lower range (44–51 pg/ml) for all groups of animals; thus, plasma E2 levels on Day 4 did not differ significantly from those on Day 0. Based on these data, we consider that all groups of animals were exposed to equivalent E2 priming.
Plasma P4 Rapidly Increases in PE Rats Given Injected P4 after OVX and E2 Implantation
Before OVX, the mean plasma levels of P4 in the EO, EP, and LP groups were 20.6 ± 3.9, 25.7 ± 5.7, and 32.1 ± 4.9 ng/ml, respectively (Fig. 1B). As explained earlier, it was necessary to have a blood sample collected right before OVX and placement of the E2 implant on Day 0. Because the sample was taken via the implanted catheter immediately after the blood vessel cannulation surgery, the relatively elevated plasma P4 most likely resulted from adrenal responses to the surgical stress and anesthesia. After OVX and E2 implantation, plasma P4 was diminished in all three groups of animals. Levels on Day 3 were 2.7 ± 0.5, 2.4 ± 0.8, and 2.9 ± 0.6 ng/ml for EO, EP, and LP rats, respectively. Similar low levels of P4 were also seen in animals on Day 4 before an s.c. P4 injection, which quickly raised plasma P4 within 2 h to 47.2 ± 10.2 and 35.4 ± 2.1 ng/ml for early PE (EP group) and long-term PE (LP group) rats, respectively. Thereafter, plasma P4 levels in both groups remained elevated (40–60 ng/ml) for at least 4 h. In contrast, an injection of the vehicle had no effect on plasma levels of P4 in early PE animals (EO group), and their circulating P4 remained consistently at low basal levels. Overall, the levels of P4 in all three groups of females on Day 0 were higher than those on Days 3 and 4 (P < 0.01). However, the P4 levels between the three groups were all similar on Days 0, 3, and 4 except between the EO and EP groups on the afternoon of Day 4.
P4 Influences Gonadotropin Secretion in Early PE Rats after OVX and E2 Priming
The data in Figure 2A show the patterns of plasma LH in E2-primed, OVX PE rats in response to an s.c. injection of P4 or vehicle. On Day 4, P4 administration at 1200 h produced a large and sustained rise in plasma P4 within 2 h and elicited an LH surge (EP group). At 1400 h, basal levels of plasma P4 were low (2.8 ± 0.46 ng/ml). Thirty minutes later, plasma LH started to increase rapidly, peaked at 1600 h (10.80 ± 2.42 ng/ml), and then gradually returned to low basal levels (1.85 ± 0.45 ng/ml) by 2130 h. This LH surge profile was remarkably similar to that of spontaneous, proestrous LH surges in cyclic rats [31]. In contrast, vehicle administration (EO group) had no effect on LH release in OVX E2-primed PE rats, and plasma LH levels remained consistently low (0.43–1.64 ng/ml) throughout the 9-h period.
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Figure 2B depicts the patterns of plasma FSH following administration of P4 or vehicle. Basal plasma levels of FSH were 381 ± 25 and 459 ± 47 ng/ml for the EO and EP groups, respectively, at 4 days post-OVX and E2 implantation. Administration of P4 to EP animals at 1200 h elicited a large increase in plasma FSH, reaching its peak level (1251 ± 178 ng/ml) by 1700 h. Thereafter, plasma FSH gradually decreased, but it remained elevated even 10 h after P4 administration. In contrast, administration of corn oil had no effect on FSH release in the EO group, and plasma FSH remained at steady levels throughout the period of sampling.
Effects of P4 on Gonadotropin Surges in Long-Term PE Rats after OVX and E2 Implantation
We subsequently compared the positive-feedback response in the LP animals to the EP group. As shown in Figure 1, the plasma patterns of E2 and P4 after OVX and E2 implantation in long-term PE rats were similar to those of the EP group of early PE females. On Day 4, the patterns of rise in plasma P4 following P4 administration at 1200 h were also similar between the early PE (EP group) and long-term PE (LP group) animals. However, under conditions of the same P4 treatment and similar plasma E2 and P4 levels, P4 challenge could only elicit discernible LH surges in four of six long-term PE females (Fig. 3). In these four responsive LP females, the peak value of their LH surges were 11.8, 11.2, 8.8, and 5.3 ng/ml, whereas the plasma LH were less than 5.0 ng/ml in the two nonresponsive LP rats (rats PR18-1 and PR15-5) (Fig. 3, bottom). In contrast to the LH response, only one of the four LH-responsive females displayed a discernible FSH surge (rat PR19-1) (Fig. 3, top).
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Notably, the LP rats that displayed an LH or FSH surge (or both) were in the acyclic PE state for less than 80 days, whereas the other two nonresponsive LP females had experienced 96 consecutive days of vaginal estrous (Fig. 3). Furthermore, in comparisons of these six LP females as a single older group versus the early PE rats (EP group), the rise in plasma LH induced by E2 and P4 in the LP animals was relatively slow and significantly lower (Fig. 4). Separate examination of the four responsive LP females versus the early PE group also support the trend that the plasma LH rise in this cohort was lower, on average, than that of the early PE animals (data not shown).
Plasma P4 in OVX, E2-Implanted, Early PE Rats before and after P4 Administration and Caging with Males
The data in Figure 5 reveal the plasma patterns of P4 in early PE female rats taken 1–4 days after OVX and E2 implantation followed by an s.c. injection of P4 or corn oil on Day 4 and after subsequent caging with males at 1400 h. Plasma concentrations of P4 remained consistently low on Days 3 and 4 in PE rats after OVX and E2 implantation (Fig. 5B). On Day 4, an s.c. injection of P4 at 1200 h greatly increased plasma P4 to a peak of 71.45 ± 15.28 ng/ml at 1530 h (EPM group) (Fig. 5C), whereas animals receiving vehicle displayed a minimal increase in plasma P4 (EOM group: 19.62 ± 4.39 ng/ml) at 1500 h, with a rapid decline to baseline levels at approximately 4–5 h.
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Effects of P4 and Caging with Males on Plasma LH and FSH Levels
Changes in plasma patterns of the gonadotropin surges in early PE females following P4 administration and caging with males are shown in Figure 6A. Four days after OVX and a sustained rise in plasma E2 because of the implant, an s.c. injection of corn oil at 1200 h was given, followed by caging with a male beginning at 1400 h. This protocol elicited a prompt, but moderate, increase in plasma LH (4.72 ± 1.51 ng/ml) within 1 h, followed by a decline to the pretreatment level within 4–5 h in the EOM group. Interestingly, when the P4-injected, early PE rats were caged with males (EPM group) 2 h later, a prompt rise in plasma LH occurred within 1 h, with a peak (10.79 ± 2.84 ng/ml) at 1600 h and then a gradual decline.
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As shown in Figure 6B, plasma patterns of FSH in the EOM group were similar to their LH levels, with EOM animals only showing a small, delayed peak in plasma FSH (671.4 ± 98.9 ng/ml) at 2 h after caging. In contrast, administration of P4 and caging with males elicited a large FSH surge (1208.6 ± 140 ng/ml) in the EPM group.
Effect of P4 on Lordosis Behavior of PE Rats
To determine the behavioral consequences of E2 priming followed by P4 challenge, the lordotic behavior of PE rats was examined (Table 2). The average number of mounts performed by the males used for mating in this study was 29.0 ± 3.1 in 3.0 min. On Day 4, PE females were introduced to males at 1400 h immediately after sampling and injection either with oil (EOM group) or with P4 (EPM group). Within 30 min of the observation period, the number of lordosis events for EOM and EPM rats was 23.6 ± 4.5 and 32.6 ± 3.6, respectively. The number of mounts by male EOM and EPM rats was 24.6 ± 4.6 and 32.9 ± 3.5, respectively. No significant difference in the numbers of lordosis, the numbers of mounts, or the lordosis quotient was found between the EOM and EPM groups; therefore, both groups of PE females displayed similar lordotic behavior. However, P4 administration shortened the time required to display the first lordosis in EPM rats (18.3 ± 7.5 min) compared with that observed in EOM rats (34.3 ± 8.2 min).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The adrenal gland provides a source of mating-induced P4, because P4 levels are present, if reduced, after OVX [15]. Therefore, we also confirmed the relevance of physiologic P4 production to elicit the LH surge and to affect lordotic behavior in additional caging/mating studies. Mating with a fertile male induced moderate increases in plasma LH and P4 of early PE animals, whereas P4 administration following caging/mating with males increased the LH secretion to a more prompt and sustained rise. Thus, circulating levels of P4 influence the timing of the gonadotropin surges and also enhance their amplitude. These observations extend those of previous studies showing a prompt and large increase in LH release by caging/mating of aging PE animals with sexually active males [17, 18, 33] and the rapid and marked increase in plasma P4 occurring concomitant with the male-induced LH surge [18, 19]. Notably, whereas caging of PE animals alone (without intromission) induced a small P4 rise but no LH surge, mating of PE animals led to a substantial P4 rise and the display of an LH surge [18, 19]. The E2 priming, followed by either P4 or corn oil administration, produced similar high degrees of lordotic behavior in our study of OVX, early PE animals. Thus, lordotic responses in early PE may be primarily under the control of E2 rather than P4. Collectively, these results support the hypothesis that mating-induced stimuli activate the neuroendocrine system to increase P4 secretion, which then facilitates LH surges via the hypothalamic-pituitary system.
The chronic elevation in E2 and absence of ovarian P4 may result in altered central nervous system functions during the prolonged PE state. The changes in the central nervous system of the age-related transition to acyclicity include a decrease in LH pulse frequency, which occurs in parallel with the increase in reproductive decline [34], and a marked delay in the increased LHß mRNA expression that usually follows OVX [35]. Reductions in P4-receptor (PR) mRNA levels also occur at sites involved in regulation of the LH surge (e.g., the anteroventral periventricular nucleus) of both early and long-term PE rats, supporting the idea of altered PR responsiveness in the hypothalamic-pituitary axis. In contrast, only long-term PE animals display reduced PR mRNA levels in the ventromedial hypothalamus and arcuate nucleus [36]. The trends in our long-term PE data may reflect the physiologic relevance of this finding given that a portion of the late PE animals either lost gonadotropin surge responsiveness to ovarian steroid stimulation after 80 days of chronic PE (e.g., long-term PE rats PR18-1 and PR15-5) (Fig. 3) or showed a diminution in the gonadotropin surge responsiveness.
These temporal changes in the positive feedback mechanism during a prolonged PE state may be age-independent, and they relate primarily to the chronic elevation in E2 and absence of ovarian secretion of P4. Chronic elevation of E2 for more than 7 wk in young OVX rats almost completely inhibits the LH surge-inducing actions of E2 plus P4 [21], whereas 4 wk of elevated E2 exposure in young OVX rats substantially suppresses E2 priming (with or without P4) of the positive feedback on the LH surge [22, 32]. Such diminished responsiveness may partly involve decreased activation of the hypothalamic GnRH neurons [37]. Moreover, the dramatic estrous cycle alterations following E2 valerate bolus also associate with morphological changes in hypothalamic neuroglial cells [38].
Initial examination of the role of P4 in ovulation described its role as a facilitator of ovulation. The s.c. P4 injection to rats hypophysectomized just after a proestrous rise in LH release could still induce ovulation as an LH substitute [39]. Subsequent reports demonstrated that additional P4 can also enhance the E2-induced surge [17, 18]. Recent studies now suggest that the role of P4 in ovulation may also extend to a counteraction of the deleterious effects of chronic E2 on the LH surge mechanism [40], because addition of P4 within 4–8 days after the start of E2 treatment can increase the proportion of young OVX rats showing LH surges. Presumably, the cessation of exposure to chronic E2 may be essential to restoration of the positive-feedback effect of E2 plus P4 on the LH surge in PE animals. This contention also agrees with conditions involving a transition from chronic E2 to P4 production, as in pseudopregnancy of rodents, when the reproductive state switches from PE to a marked elevation of P4 concomitant with diminished E2 [16]. Following pseudopregnancy, an aging animal can spontaneously resume the positive-feedback mechanism, yet how these circumstances lead to responsiveness requires further study.
The hypothesis of chronic E2 damage in the absence of P4 may also describe how termination of chronically elevated E2 in PE animals reinstates responsiveness of the hypothalamic-pituitary axis. Placement of E2 implants in young OVX rats resulted in daily afternoon LH surges [32, 41], but similar treatment in long-term PE females did not lead to an LH surge [17]. Moreover, P4 challenge after E2 priming also failed to elicit an LH surge in acutely OVX PE rats [16, 21]. However, at 5 wk following OVX in either PE or young rats, administration of E2 with P4 will induce an LH surge in both groups, although the magnitude of the LH surge induced is lower in the OVX PE rats compared with that in the OVX young rats [21].
Presently, no conclusive evidence elucidates the causal relationship between loss of the LH surge and onset of PE. Based on our observations, we propose the following sequence for the neuroendocrine control of the gonadotropin surges during reproductive senescence: In young animals, the cyclic increase in E2 secretion is required to elicit the proestrous LH and FSH surges, whereas the rise in plasma P4 associated with the initial LH surge may facilitate these neuroendocrine events. By middle age, the magnitude of the preovulatory LH surge on the afternoon of proestrus is attenuated [8–11, 42] even when these animals show a regular cycle. This attenuation in LH secretion is a hallmark of neuroendocrine aging, and it occurs close to the cessation of regular cyclicity. The prolonged and extended estrous phase of an irregular cycle indicates chronically elevated E2 and reduced P4 in the circulation of a middle-aged female rat, and this hormonal milieu is unfavorable for normal expression of the positive feedback on LH secretion [17]. This hypothesis may explain why E2 plus P4 either fails to elicit gonadotropin surges in long-term PE animals or induces a lesser gonadotropin surge response (Fig. 3) relative to that of early PE animals [17, 36]. Thus, the results of the present study, in combination with those of previously published work, demonstrate that E2 positive feedback is not lost until the middle-aged animal enters the acyclic PE state. In early PE females, E2 plus P4 can elicit gonadotropin surges, whereas E2 alone cannot. Our evidence further verifies that such a resistance of the hypothalamic-pituitary axis to the surge-inducing ability of E2 has occurred soon after the aging rat enters a PE state (i.e., early PE). These results suggest that exposure of young rats to chronic E2 causes a sequential loss of the stimulatory actions first of E2 and then of P4. Presumably, the continued exposure to high E2 and low P4 for the 3–4 mo during long-term PE additionally suppressed the ability of E2 plus P4 to induce an LH surge. However, additional study of the long-term PE state is necessary to confirm this trend. Therefore, the results of the present study support the hypothesis that onset of the PE state in the female rat is associated with a loss of the positive effect of E2 on LH secretions, whereas the neuroendocrine system during the initial phase of the PE state still responds to the facilitative action of P4 on the LH surge. Further investigation into the early PE state will be necessary to determine the specific mechanisms through which P4 maintains neuroendocrine responsiveness.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: John K.H. Lu, Division of Reproductive Endocrinology, Department of Obstetrics & Gynecology, CHS 22-172, D. Geffen School of Medicine at UCLA, 10833 Le Conte Avenue, Los Angeles, CA 90095-1740. FAX: 310 206 3670; jlu{at}mednet.ucla.edu
3 Current address: Department of Internal Medicine, Division of Endocrinology & Metabolism, University of Virginia, Charlottesville, VA 22908-0578
4 Current Address: Department of Biological Sciences, California State University Los Angeles, Los Angeles, CA 90032
Received: 27 February 2004.
First decision: 19 March 2004.
Accepted: 14 April 2004.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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