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Biology of Reproduction 64, 1056-1062 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Influences of Age and Ovarian Follicular Reserve on Estrous Cycle Patterns, Ovulation, and Hormone Secretion in the Long-Evans Rat1

Christopher R. Anzalonea, Long-Sheng Hongb, John K.H. Lub, and Philip S. LaPolt2,6,b

a Departments of Obstetrics & Gynecology, Biology, and Neurobiology and b the Laboratory of Neuroendocrinology of the Brain Research Institute, University of California, Los Angeles, Los Angeles, California 90095

ABSTRACT

This study examined the influences of aging and reduced ovarian follicular reserve on estrous cyclicity, estradiol (E2) production, and gonadotropin secretion. Young virgin and middle-aged (MA) retired breeder female rats were unilaterally ovariectomized (ULO) or sham operated (control). Unilateral ovariectomy of young rats reduced the ovarian follicular reserve by one-half, to a level similar to that found in MA controls. Unilateral ovariectomy of MA females reduced the follicular pool further, to one half of MA controls. The incidence of regular cyclicity was significantly lower in MA ULO females than in young controls, with intermediate cycle frequency in young ULO and MA controls. Among cyclic rats, the magnitude of the proestrous LH surge was highest in young controls, intermediate in young ULO rats and MA controls, and lowest in MA ULO females. Similarly, ovulation rates were highest in young controls, intermediate in young ULO rats and MA controls, and lowest in MA ULO females. While young ULO rats exhibited augmented secondary FSH surges on estrous morning, middle-aged ULO females displayed secondary FSH levels comparable to young controls. The effects of age and reduced follicle number on estrous cyclicity and gonadotropin secretion were not due to altered E2 secretion, as preovulatory E2 levels were similar among all groups. Thus, experimental reduction in the follicular reserve exerts acute effects on the preovulatory LH surge, ovulation rate, and estrous cyclicity in both young and MA rats. However, decreased follicle number increases FSH levels only in young rats, indicating aging-related alterations in the feedback regulation of FSH.

aging, estradiol, FSH, follicle, LH, ovary, ovulation, ovulatory cycle

INTRODUCTION

In the female rat, reproductive aging is characterized by gradual declines in regular estrous cyclicity and fertility at middle age [1–6]. These reproductive declines are associated with gradual impairments in ovulatory function [7] and altered patterns of gonadotropin and steroid secretion [8–13]. A hallmark of reproductive aging in the female rat is the attenuation of the preovulatory LH surge on proestrus that serves as a marker for neuroendocrine aging and the imminent loss of regular estrous cycles [8, 9, 11]. While alterations in hypothalamic functions have been demonstrated in aging female rats [14–16], it is unclear whether the decline in LH surge magnitude is due primarily to deficits in neuroendocrine function, altered ovarian function, or both. Because the preovulatory rise in circulating estradiol (E2) serves as the functional trigger for the LH surge, age-related changes in E2 production may contribute to decreased gonadotropin release during the afternoon of proestrus. However, the potential role of altered E2 levels in impaired neuroendocrine function is not clear.

Concomitant with the proestrous LH surge, there is a GnRH-dependent preovulatory surge of FSH on the afternoon of proestrus, followed by a GnRH-independent rise in FSH starting early on estrous morning, referred to as the secondary FSH surge [17, 18]. The magnitude and duration of the secondary FSH surge is believed to be regulated by circulating levels of the gonadal peptide inhibin, produced from developing follicles [19–21]. The secondary FSH surge is thought to play an important role in the selection of small antral follicles that will continue growth and potentially reach the preovulatory stage on the following proestrus [22–25]. While some investigations [12, 13] have reported an augmentation or prolongation of the secondary FSH surge in middle-aged (MA) rats (possibly due to decreased ovarian follicular reserves), others report no significant change in FSH levels with age [11]. Interestingly, unilateral ovariectomy (ULO) of young rats increases the magnitude of the secondary FSH surge, associated with compensatory ovarian hypertrophy and the maintenance of normal ovulation rates [26]. These findings in MA intact and young ULO rats suggest that there are interactions between follicular pool size, the numbers of developing follicles on estrus, and the magnitude of the secondary FSH surge.

In the rat, reproductive aging is associated with a reduced ovarian follicular reserve [27]. It has been demonstrated that slowing the age-related decline in follicular number by caloric restriction [28] or successive progesterone treatments [29] can concomitantly delay the loss of regular estrous cyclicity as well as maintain fertility to an advanced age. Furthermore, surgically reducing ovarian follicular reserves in young rats accelerates reproductive impairments [30, 31]. Hence, the age-related decline in follicular number may play an integral role in the ovarian and neuroendocrine alterations that contribute to reproductive dysfunction during aging. However, the specific impact of a decreased ovarian follicular pool on altered neuroendocrine and steroidogenic function has not been examined. To elucidate further the influences of aging and of decreased ovarian follicular reserve on neuroendocrine and ovarian functions, the present study examined the effects of chronological age and unilateral ovariectomy on the maintenance of estrous cyclicity, circulating E2 level, preovulatory LH surge magnitude, ovulation rate, and secondary FSH surge in young and MA intact and ULO female rats.

MATERIALS AND METHODS

Animals

Young (90–100 days) virgin and MA (8–9 mo) retired breeder Long-Evans female rats were received from Charles River Laboratories (Portage, MI). Animals were 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. Food and drinking water were available ad libitum. Daily vaginal smears were obtained from both young and MA rats to monitor individual estrous cycle patterns, and only regularly cyclic rats were used in this study. Experiments were conducted in accordance with the International Guiding Principles for Biomedical Research Involving Animals as promulgated by the Society for the Study of Reproduction.

Methods

Influences of unilateral ovariectomy on estrous cyclicity and gonadotropin secretion in young and MA rats To assess the influence of ovarian follicular reserve on reproductive function, young and MA rats displaying at least two consecutive 4-day-long estrous cycles were either ULO or sham operated, and their subsequent estrous cyclicity and circulating gonadotropin levels were compared. At 1200 h on proestrous, ULO or sham surgery was performed while the animal was under light ether anesthesia. Following ULO, some animals continued to display regular cycles, while others exhibited irregular estrous cyclicity associated with extended days of vaginal estrus. Those animals that remained regularly cyclic for three consecutive 4-day cycles were implanted with jugular vein catheters to allow frequent blood sampling. Cannulation was performed late in the afternoon on diestrous Day 2, using the method of Harms and Ojeda [32], with some modification [11]. On the next day (i.e., 12 days after ULO or sham surgery), proestrous females were serially sampled for gonadotropin measurements. During 1400–2130 h on proestrus, sequential blood samples (0.1 ml each) were taken via the catheter every 90 min to characterize the magnitude of the proestrous LH surge. Samples were collected into heparinized syringes and immediately centrifuged, and the plasma stored at -20°C until assayed for LH. To characterize the secondary FSH surge in these same animals, additional blood samples (0.15 ml each) were similarly obtained at 2400 h on proestrus, 0200 h on estrus, and once every 90 min from 0400–1300 h on estrus, and the plasma was separated and stored until assayed for FSH.

Ovulation rates and numbers of developing follicles in young and MA ULO and intact rats After the last blood sample at 1300 h on estrus, each animal was anesthetized with ether and killed by cervical dislocation. The oviducts were excised, placed onto a watch glass and flushed with saline. Under a dissecting microscope, the contents of the oviducts were examined and the numbers of tubal ova counted. In addition, ovaries were dissected out and immediately placed in Bouin solution fixative for 24 h, followed by subsequent dehydration in increasing concentrations of ethanol [33]. Ovaries were then embedded in paraffin and serially sectioned at a thickness of 7 µm, followed by staining with hemotoxylin and eosin. Each ovary was coded to blind the observer, and every sixth section was examined under a microscope at 400x magnification to count the number of resting follicles (primordial and small primary follicles <50 µm in diameter), which we have previously shown to be related to the onset of reproductive aging [29]. The total number of resting follicles was obtained by multiplying the sum of the counted numbers by six, similar to the method of Faddy et al. [34]. Also, the numbers of nonatretic developing follicles (>100 µm in diameter) were also similarly determined under 100x magnification. Developing follicles containing two or more granulosa cells with pyknotic nuclei were considered to be atretic [35].

Plasma patterns of E2 during the estrous cycle in ULO and control rats To determine the effects of ULO and chronological age on E2 secretion, separate groups of young control and MA control and ULO rats were prepared as described above and their estrous cycle patterns were monitored. Only those animals that maintained two consecutive 4-day estrous cycles after surgery were used in this experiment. On diestrous Day 2 of the second estrous cycle (i.e., 7 days after surgery), each animal received an indwelling jugular catheter as described above. Beginning at 1800 h on diestrus Day 1 of the third cycle (i.e., 10 days after surgery) and continuing until 1800 h on proestrus, sequential blood samples (0.5 ml each) were taken every 6 h via the catheter for subsequent assay of plasma E2. To reveal the patterns of LH secretion in these same individual rats, additional blood samples (0.1 ml each) were also taken every 90 min during 1400–2130 h on proestrus for subsequent assay of plasma LH. At 0900–1000 h the next morning, animals were killed and the oviducts were flushed to determine the numbers of tubal ova, as described above.

Hormone assays Plasma concentrations of LH and FSH were measured by double-antibody RIA methods, employing the reagents distributed by the National Hormone and Pituitary Program, NIDDK, NIH, as previously described [11]. The LH and FSH values are expressed in terms of the reference standards, rat LH-RP-1 and rat FSH-RP-1, NIDDK, 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. Plasma levels of E2 were measured by specific RIA as previously reported [6]. Prior to RIA, plasma samples were extracted with diethyl ether, and the E2 fraction was isolated by Celite (Celite Co., Lompoc, CA) column chromatography [36]. The intra- and interassay coefficients of variation for the E2 RIA were 5.4% and 6.9%, respectively.

Statistical analyses Comparisons of the follicular pool sizes, ovulation rates, and plasma hormone levels between the treatment groups were performed by two-way ANOVA, followed by Student-Newman-Keuls test when appropriate. The incidences of regular cyclicity in each group were compared by chi-square test. Correlations between the follicular pool size and plasma hormone levels as well as between the numbers of resting and developing follicles were performed by linear regression analysis using the Pearson's product moment test. A confidence level of P < 0.05 was considered statistically significant.

RESULTS

Influence of Age and ULO on the Ovarian Follicular Reserve and Number of Developing Follicles

Young and MA animals were ULO or sham-operated (control) and subsequently sampled to characterize hormone levels. From these same animals, the ovaries were removed for histological processing and examination. Figure 1 depicts the total numbers of resting follicles (primordial and small primary follicles) among the four groups. In young rats, the total number of resting follicles was found to be 5887 ± 449 in control females (n = 16), significantly higher than that in the other groups (P < 0.001). Young ULO animals (n = 19) had approximately half as many resting follicles (3260 ± 270 follicles/rat) as young intact rats. Interestingly, young ULO females had similar numbers of resting follicles as MA control rats with two ovaries (3004 ± 485 follicles/rat; n = 17). In MA ULO animals (n = 17), the number of resting follicles (1693 ± 249 follicles/rat) was significantly lower (P < 0.01) than that in the other three groups. Hence, the follicular pool size was greatest in young intact females, intermediate in young ULO rats with one ovary and MA intact animals with two ovaries, and lowest in MA ULO females.



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  		FIG. 1. Mean numbers of resting follicles (<50 mm in diameter, mean ± SEM) in ovaries of young and MA intact and ULO animals (n = 16–19/group). Groups with different letters above bars are significantly different from each other (P < 0.05)

Figure 2 shows the numbers of nonatretic developing follicles in the ovaries of young and MA rats on estrus. Young control females had a total of 208 ± 9 developing follicles in two ovaries, significantly greater than any of the other three groups (P < 0.05). Young ULO rats had 147 ± 8 developing follicles in one ovary, similar to the total numbers of developing follicles in both ovaries of MA intact rats (157 ± 22). Middle-aged ULO animals had the lowest numbers of developing follicles on estrus (80 ± 9), significantly less than that observed in any of the other three groups (P < 0.05). Linear regression analysis revealed a significant correlation between the numbers of resting and developing follicles in individual rats of all groups (r = 0.74; P < 0.001).



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  		FIG. 2. Mean numbers of nonatretic developing follicles (>100 µm diameter) present at 1300 h on estrus in young and MA, intact and ULO rats (n = 16–19 animals/group). Significant differences between groups (P < 0.05) are indicated by different letters above bars

Influence of Age and Reduced Follicle Number on the Maintenance of Estrous Cyclicity

After sham or ULO surgery, estrous cycle patterns were monitored in young and MA animals by daily vaginal lavage. The incidence of regular cyclicity after surgery was highest in young control rats (83%, n = 30), significantly greater (P < 0.05) than that in MA control (58%, n = 52) and MA ULO (45%, n = 65) females (Fig. 3). The incidences of regular cyclicity in young ULO (73%, n = 40) and MA controls were not significantly different, whereas MA ULO rats had a lower incidence of regular cycles than young ULO and young control rats (P < 0.05). These findings indicate influences of both age and ovarian follicular reserve on the maintenance of regular estrous cyclicity.



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  		FIG. 3. Incidence of regular cyclicity (percentages of rats completing three regular 4-day-long estrous cycles immediately after ULO or sham surgery) in young and MA females

Influence of Aging and Reduced Follicle Number on Proestrous LH Surge Magnitude

The magnitude of the proestrous LH surge was characterized in young and MA intact and ULO females during the third estrous cycle after surgery (Fig. 4). In all groups, an increase in LH release began around 1530 h and peaked by 1830 h. Young controls (n = 17 rats) displayed high preovulatory LH surge levels, with a value at 1830 h of 1210 ± 115 ng/ml. In contrast, LH surge magnitudes in MA ULO rats were significantly attenuated, reaching levels of only half that seen in young controls (P < 0.05, n = 17). Both young ULO and MA control rats displayed LH surges that appeared lower than those of young controls, but these differences were not statistically significant. We also determined the average peak LH value of animals in each group, to correct for differences in the timing of the LH surge between individual animals (Fig. 5). This analysis revealed that peak LH values were significantly lower in MA intact and MA ULO animals than in young intact females. In addition, peak LH levels were lower in MA ULO rats than young ULO females (Fig. 5).



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  		FIG. 4. Proestrous LH surge levels in young and MA intact and ULO rats (n = 16–19 animals/group). The LH surge profiles are significantly different (P < 0.05) between young intact and MA ULO females



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  		FIG. 5. Peak proestrous LH surge values for young and MA intact and ULO rats (n = 16–19 animals/group). Groups with different letters above bars are significantly different from each other (P < 0.05)

Linear regression analysis revealed a significant correlation between the peak LH surge value and the numbers of resting follicles in young animals (r = 0.64; P < 0.01) and in MA rats (r = 0.42; P < 0.05), such that female rats with high follicular reserves tended to have high peak LH values, and those with few resting follicles exhibited lower LH surge magnitudes.

Influences of Age and Reduced Follicle Number on Secondary FSH Surge Magnitude

Following sampling to characterize the LH surge, serial blood samples were also collected from 0000 h to 1300 h on estrus in these same animals in order to characterize secondary FSH surge profiles. Figure 6 depicts FSH levels in the four groups of rats, and only those animals that displayed LH surges followed by ovulation were included in data analysis. All four groups of rats displayed a secondary FSH surge between 0300 to 0900 h on estrous morning. In young animals, the secondary FSH surge was significantly higher (P < 0.05) in ULO females than in control rats. The FSH levels were markedly elevated in young ULO rats as early as midnight proestrous. In MA controls FSH levels were slightly, but not significantly, elevated compared with young controls. Surprisingly, ULO of MA rats did not further enhance the secondary surge of FSH on estrus. Thus, decreasing the ovarian follicular reserve augments FSH release on estrus in young but not MA rats.



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  		FIG. 6. Secondary FSH surge levels in young and MA intact and ULO rats. Young ULO females displayed significantly elevated secondary FSH surges (P < 0.05) compared to young intact rats

Influence of Age and Reduced Follicle Number on Ovulation Rates

Following the termination of blood sampling at 1300 h on estrus, the oviduct contents were examined to determine ovulation rates in these same animals. Young intact rats (n = 17) that exhibited high LH surges had significantly (P < 0.05) higher ovulation rates (13.2 ± 1.3 ova/rat) than MA ULO animals (6.8 ± 1.9 ova/rat, n = 17) (Fig. 7). Young ULO (n = 19) and MA intact rats (n = 17) displayed ovulation rates (9.9 ± 1.4 ova/rat and 8.9 ± 1.9 ova/rat, respectively) that were not statistically different than those of young intact or MA ULO females. Thus, marked reduction in the follicular reserve in MA rats resulted in a significant decrease in ovulatory function.



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  		FIG. 7. Ovulation rates (mean ± SEM) of young and MA intact and ULO animals (n = 16–19/group). Groups with different letters above bars are significantly different from each other (P < 0.05)

Effects of Age and Reduced Follicle Number on Plasma E2 Patterns

Because peak LH levels were significantly lower in MA intact and ULO rats compared with young intact females, we characterized the preovulatory rise in circulating E2 in different groups of young (n = 7) controls, as compared to MA controls (n = 11), and MA ULO females (n = 14) (Fig. 8) to determine if attenuated LH levels in MA rats were associated with decreased E2 production. All three groups exhibited a marked increase in E2 secretion beginning on the afternoon of diestrous Day 2, reaching the peak level on the early afternoon of proestrus. No significant differences in plasma E2 were found between the three groups. The LH surge levels were characterized in these same rats, and, as in the first group of animals, MA ULO females demonstrated significantly attenuated surges when compared to young intact rats (data not shown) despite normal patterns of circulating E2.



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  		FIG. 8. Mean levels of circulating estradiol in young and MA intact (n = 7 and 11, respectively) and MA ULO rats (n = 14). Blood sampling began at 1800 h on diestrous-1 and ended at 0930 h on the following estrus

DISCUSSION

Middle-aged female rats experience significant changes in neuroendocrine and ovarian functions, associated with the loss of regular estrous cyclicity and decreased fertility and fecundity. While these changes are temporally related to a decline in ovarian follicle reserves, the impact of reduced follicular pool size on neuroendocrine and ovarian functions during aging has not been previously assessed. Results from the present study clearly indicate that both age and reduced ovarian follicular reserve exert significant influences on the neuroendocrine system and on the ovary.

Previous studies by Butcher and colleagues demonstrated that experimental reduction of the follicular reserve advanced age-related declines in regular cyclicity and reproductive function [30, 31]. Conversely, augmenting the follicular pool of MA mice by transplantation of young ovaries restores ovulatory function and gonadotropin secretion [37]. Our results confirm an influence of follicular pool size on the maintenance of estrous cyclicity that may be related to alterations in LH surge magnitude. Diminished or absent LH surges on proestrous may lead to lengthened estrous cycles or the complete cessation of cyclicity. Our findings suggest that reduction of the follicular reserve has a significant impact on the preovulatory LH surge magnitude during the evening of proestrous. The LH surge magnitude was significantly correlated with the numbers of resting follicles in young and MA, intact and ULO rats. While LH levels were not different between young ULO and MA intact rats, further decreasing the follicular reserve of MA rats by ULO resulted in significantly lower peak LH levels than in young ULO animals. These findings suggest a neuroendocrine contribution to the declines in cyclicity associated with follicular reduction. In addition, ovulation rates were decreased in MA ULO rats, possibly reflecting decreased proestrous LH magnitudes [38–40] or decreased numbers of preovulatory follicles available for rupture.

It is also clear from our data that factors in addition to follicular number influence the parameters studied. While young ULO and MA intact rats have similar follicular reserves, only MA intact rats displayed a decreased incidence of regular cyclicity and lower peak LH levels compared with young intact females. Similarly, while young ULO females exhibited an augmented secondary FSH surge compared to young intact rats, no difference in FSH levels was observed in MA intact females. Presumably, these differences reflect differences in age between young ULO and MA intact rats. However, it should also be noted that paracrine interactions in ovaries of ULO animals undergoing compensatory hypertrophy may differ from those in intact animals, potentially resulting in altered ovarian function, in addition to a decreased follicular reserve. In addition, the reproductive history of the groups studied differed, in that young animals were nulliparous and MA animals were multiparous. While multiparous animals undergo reproductive aging more slowly than virgin animals [7], any effects of this difference on the specific parameters studied are not known.

The precise mechanisms resulting in diminished LH surges during aging or reduction of the follicular reserve are unclear. During the estrous cycle, increasing levels of E2 from developing follicles act as a trigger to elicit the LH surge on proestrus. Therefore, we hypothesized that attenuated LH surges displayed by MA intact and ULO rats may be related to decreased numbers of developing follicles and associated declines in E2 production. Similar to previous studies, we report a direct correlation between the size of the ovarian follicular reserve and the numbers of developing follicles observed on estrous morning [41, 42]. However, our data indicate that there is no effect of age or reduced follicular number on E2 production during the estrous cycle. Both young and MA intact, as well as MA ULO regularly cyclic animals display similar patterns of E2 release throughout their estrous cycles. Furthermore, even those rats that failed to exhibit proestrous LH surges (presumably those that would become irregularly cyclic) had E2 profiles that were similar to those of young intact females (data not shown), suggesting impaired neuroendocrine response to E2.

The findings from the present study also demonstrate that while reduction of follicular number in young rats results in a compensatory increase in FSH secretion on estrous morning, ULO of MA females has no effect. The secondary surge of FSH on estrous morning is believed to be important in regulating the recruitment of developing follicles [22–25]. Thus, increased FSH release in young ULO rats is associated with increased follicular development, such that the number of preovulatory follicles in the remaining ovary of a ULO female is similar to the combined number in both ovaries of young intact rats [42]. There have been conflicting reports as to whether MA rats experience augmented secondary FSH surges. While DePaolo and colleagues have reported increased FSH secretion on estrus in MA rats [10, 12], we have not observed statistically significant increases in circulating FSH in aging animals in this and previous studies [11]. Butcher and colleagues have demonstrated that experimental reduction of follicle number in MA rats increases basal FSH release on diestrous Day 1, they did not examine the effects of ULO on the secondary FSH surge [30, 31]. In any event, the present study clearly demonstrates that further decreasing follicle number by ULO in MA rats also fails to augment the secondary FSH surge, indicating that the compensatory response in FSH secretion on estrus observed in young ULO females does not occur in aging rats.

The mechanism(s) by which MA rats maintain relatively normal secondary FSH surge levels, despite decreased follicle number, are not known. One major factor believed to regulate secondary FSH surge magnitude is the levels of circulating inhibin [19–21, 23, 25, 43, 44]. Limited data are available regarding the effects of a decreased follicular reserve on dimeric inhibin levels early on the morning of estrous. There is one report of decreased inhibin bioactivity on estrous morning in MA rats, associated with elevated secondary FSH surge levels [12]. Conversely, ovarian inhibin {alpha} subunit mRNA levels are reportedly higher in middle-aged rats on estrous morning, compared to young cyclic females [45]. Our preliminary data indicate that ULO decreases circulating dimeric inhibin levels and ovarian inhibin subunit expression in young but not MA rats, compared with young intact animals [46]. Thus, the failure of ULO to augment the secondary FSH surge in MA rats may reflect altered regulation of inhibin expression. In addition, it is not known whether age-associated deficits in pituitary function contribute to impaired FSH release. Further work is required to examine the effects of aging and reduced follicular reserve on the mechanisms regulating the secondary FSH surge.

One confounding factor in interpreting these data is the fact that the greatest differences among groups were found between young intact and MA ULO females. These groups have the greatest variance in follicle reserve, in addition to their age difference. Thus, it is not clear whether the large differences found between these two groups are due to highly diminished follicular reserves of MA ULO animals alone or to an interaction between follicular reserve and age. As described above, there were more differences in LH secretion, estrous cyclicity, and ovulation rate between young intact and MA ULO than young intact and MA intact rats, indicating an effect of ULO on these functions in MA animals. Whether there is also a contribution of chronological age to these differences is assumed but not known. Practical considerations of manipulating a young animal such that its follicular reserve is similar to that of MA ULO rats make such experimental comparisons of young and MA rats difficult.

The current study demonstrates influences of chronological age and ovarian follicular pool size on reproductive senescence. Our data suggest that age-related changes in neuroendocrine and ovarian functions reflect diminished follicular reserves in addition to well-documented changes in the hypothalamic regulation of gonadotropin release.

ACKNOWLEDGMENTS

The authors thank the National Hormone and Pituitary Program, NIDDKD, NICHD, and the United States Department of Agriculture for the generous gift of gonadotropin RIA reagents.

FOOTNOTES

First decision: 16 October 2000.

1 This study was supported by NIH grants AG04810 and AG10620, awarded by the National Institute on Aging. C.R.A. was a Predoctoral Trainee supported in part by a Supplement to NIH grant AG04810. Back

2 Correspondence: Philip S. LaPolt, Department of Biology & Microbiology, 5151 State University Drive, California State University, Los Angeles, CA 90032. FAX: 323 343 6451; plapolt{at}calstatela.edu Back

Accepted: November 7, 2000.

Received: September 14, 2000.

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