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BOR - Papers in Press, published online ahead of print March 2, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.038745
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BIOLOGY OF REPRODUCTION 73, 45–53 (2005)
DOI: 10.1095/biolreprod.104.038745
© 2005 by the Society for the Study of Reproduction, Inc.

Bovine Model for the Study of Reproductive Aging in Women: Follicular, Luteal, and Endocrine Characteristics1

Pritpal S. Malhi , Gregg P. Adams , and Jaswant Singh 2 

Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Canada S7N 5B4


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
At present, there is no well-characterized animal model to study the effects of aging on fertility in women. The objectives of the study were to characterize age-related changes in ovarian and endocrine functions in old cows and to investigate the validity of a bovine model for the study of human reproductive aging. We tested the hypotheses that aging in cattle is associated with 1) elevated concentrations of gonadotropins and reduced concentrations of steroid hormones in systemic circulation and 2) increased recruitment of ovarian follicles during wave emergence. Daily ultrasonography was performed in 13- to 14-yr-old cows (n = 10) and their 1- to 4-yr-old daughters (n = 9) for one interovulatory interval to study ovarian function. Plasma samples were obtained every 12 h for determination of FSH, LH, progesterone, and estradiol concentrations. Circulating FSH concentrations were higher (P = 0.009) during follicular waves in old cows than in their daughters, but the number of 4- to 5-mm follicles recruited into a wave was lower (P = 0.04) in old cows. Plasma LH concentrations did not differ between groups (P = 0.4), but the ovulatory follicle in two-wave cycles was smaller in old cows (P = 0.04). Plasma estradiol concentrations were higher (P = 0.01) in old cows, and luteal phase progesterone tended to be lower (P = 0.1). We conclude that these changes are consistent with those reported for women approaching menopause transition. Therefore, our results validate the use of the bovine model to study reproductive aging in women.

aging, follicle-stimulating hormone, follicular development, ovary, steroid hormones


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Decreased fertility with maternal aging has been well documented in humans and animals [1, 2]. Infertility in older women has been associated with a dwindling ovarian follicular pool, altered hormone secretions, decreased conception rate, meiotic abnormalities, and increased gestational attrition [3–8]. Circulating concentrations of FSH in women increased in late reproductive stages [4, 6, 7], and the increase has been attributed to reduced inhibin B secretion [4, 6–10]. Steroid hormone concentrations in older women did not show a consistent pattern but eventually decreased with advanced age [4, 7, 9]. Reproductive aging in women has also been associated with a shortened follicular phase and, thus, a shortened menstrual cycle [4, 11]. The exact cause-and-effect relationships between endocrine and follicular changes and their impact on fertility remain unclear. To obviate the ethical and practical constraints that limit observational and interventional studies in humans, an animal model is required to study age-related infertility in women. To date, however, a well-characterized animal model for reproductive aging in women has not been established.

Ovarian follicular wave development in cattle has been characterized in detail over the last two decades [12], and the bovine model [13] was the foundation for the recent discovery of follicular wave development in women [14, 15]. A follicular wave has been defined as the synchronous growth of a group of follicles stimulated by a surge of FSH [15, 16]. One follicle in each wave is selected to become dominant, while others (subordinates) regress [15, 17–20]. There are two or three waves of follicular development in the majority of bovine estrous cycles [12, 17], and recent data suggest that the majority of menstrual cycles in women are also composed of two or three follicular waves [15]. Dominant follicles of waves occurring during the luteal phase are anovulatory as a result of suppression of circulating LH by progesterone [17]. Although not critically tested, results of recent studies in women [14, 15] are also consistent with the notion that progesterone secretion during the luteal phase inhibits LH release and is responsible for the ultimate regression of the dominant follicle of anovulatory waves. Demise of the previous dominant follicle, in turn, permits the emergence of a new follicular wave, and the pattern repeats itself [17, 21]. During luteolysis, decreasing concentrations of progesterone relieve suppression of LH release from the pituitary and LH-pulse frequency increases. In response, the extant dominant follicle produces increasing amounts of estradiol, which, after reaching a threshold level, is responsible for eliciting the preovulatory LH surge followed by ovulation [17, 21]. Follicular wave emergence in women, the number of waves during the menstrual cycle, dominant follicle selection, and ovulation of a single follicle were found to be fundamentally similar to ovarian patterns in cattle [13, 15] and provide justification for proposing the use of a bovine model to study ovarian function in women [13].

In one study, the mean life expectancy in cattle was 19 yr and 55% of the herd was infertile by 13 yr of age [1]. Data from another study [22] indicate that serum concentrations of FSH during Days 6–12 of the estrous cycle and preovulatory estradiol appeared to be elevated in 13-yr-old cows [22], but this study was limited to hormonal profiles and data were not studied in relation to follicular development. Thus, we expected changes in endocrine, follicular, and luteal function of 13- to 14-yr-old cows used in this study to be analogous to women approaching menopause.

The objectives of this study were to characterize age-related temporal changes in follicular, luteal, and endocrine functions and to investigate the validity of old cows as a physiological model for human reproductive aging. We tested the hypotheses that aging in cattle is associated with 1) elevated concentrations of gonadotropins and reduced concentrations of steroid hormones in systemic circulation as reported in aging women and 2) increased recruitment of ovarian follicles during wave emergence as a result of elevated circulating concentrations of FSH.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
The experiment was conducted on nonlactating, nonpregnant, crossbred Hereford cows (13–14 yr old, n = 10) and their daughters (1–4 yr old, n = 10) during the months of June and July. The cows were at least 45 days postpartum and had a corpus luteum as determined by initial ovarian ultrasonography. Animals were maintained in single outdoor corrals at Goodale Research Farm, University of Saskatchewan. The experimental protocol was approved by the University Committee on Animal Care and Supply under the guidelines of the Canadian Council on Animal Care.

Ovulation Synchronization

Ovulation was synchronized among cows using an estradiol and progesterone treatment protocol [23, 24]. Estradiol-17ß (5 mg; Catalog # E8875; Sigma Chemical Company, St. Louis, MO) and progesterone (100 mg; Catalog # P0130; Sigma Chemical Company) were dissolved in benzyl alcohol (0.4 ml; Catalog # B27354; BDH Inc., Toronto, ON, Canada), mixed with canola oil (2 ml), and given intramuscularly. An intravaginal progesterone-releasing device containing 1.9 g of progesterone (CIDR-B; Bioniche Animal Health Canada Inc., Belleville, ON, Canada) was inserted at the time of steroid treatment and was maintained in place for 7 days. On the day of CIDR removal, a luteolytic dose of prostaglandin analogue was given intramuscularly (Cloprostenol, 500 µg; Estrumate; Schering Canada Inc., Pointe-Claire, PQ, Canada). To synchronize the preovulary LH surge and ovulation, 1 mg of estradiol-17ß in canola oil was given intramuscularly 24 h after prostaglandin treatment [23].

Ovarian Ultrasonography

Transrectal ovarian ultrasonography was performed daily by the same operator using a B-mode ultrasound scanner with a 7.5-MHz linear-array transducer (Aloka SSD-900; Instruments for Science and Medicine, Vancouver, BC, Canada). Ultrasound examinations were initiated on the day of CIDR insertion to record follicular and luteal (CL) development for one complete interovulatory interval (IOI: defined as the period between two consecutive ovulations). Ovarian sketches were made during each examination to record the size and relative location of the CL and follicles ≥4 mm in diameter. Follicle diameter was recorded as the average of antral size measured in two perpendicular planes [15, 25]. The diameter of the CL was recorded similarly. The total numbers of follicles ≥2 mm were also counted in both ovaries.

Plasma Sampling and Hormone Assays

Blood samples from the jugular vein were obtained every 12 h (600 and 1800 h) in 10-ml heparinized tubes (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ) and centrifuged at 1500 x g for 15 min. The plasma was harvested and stored at –20°C. To compare characteristics of LH-pulse frequency and amplitude during expected high- and low-progesterone phases, serial blood samples were collected every 15 min for 8 h from a jugular catheter (5 ml; n = 6 cows/age group) on Days 8 and 18 of the IOI (Day 0 = ovulation). A jugular catheter (inner and outer diameters 1.0 and 1.5 mm, respectively) was fixed in place 1 day before frequent blood sampling to minimize the effects of handling stress on plasma gonadotropin concentrations.

Plasma FSH concentrations were measured using NIAMDD-anti-ovine FSH-1 primary antibody and expressed as USDA bovine FSH-I-l units [26, 27]. The range of the standard curve was 0.13–16 ng/ml, with a minimum detection limit of 0.13 ng/ml (zero ligand vs. 0.13 ng/ml, unpaired t-test, P < 0.05) [28]. The intra- and interassay coefficients of variation were 6% and 10% for low reference samples (mean, 1.68 ng/ml) and 13% and 8% for high reference samples (mean, 3.82 ng/ml), respectively. Plasma samples from mother-daughter pairs were analyzed in the same assay to distribute interassay variation equally between groups.

LH concentrations were expressed as NIDDK-bLH4 units [26, 27]. The range of the standard curve was 0.06–8 ng/ml, with a minimum detection limit of 0.06 ng/ml. The intra- and interassay coefficients of variation for LH were 10% and 5% for low reference samples (mean, 0.37 ng/ml) and 5% and 4% for high reference samples (mean, 0.97 ng/ml), respectively. The PC-Pulsar program (Gitzen and Ramirez, University of Illinois, IL) was used to characterize LH pulsatility in serial plasma samples. Pulses were identified using standard-deviation criteria of height and duration [27]. LH-pulse frequency, pulse amplitude, means, and basal concentrations were also calculated [27].

Plasma progesterone concentrations were determined using a solid-phase radioimmunoassay [29] (catalog # TKPG5; Coat-A-Count; Diagnostics Products Corporation, Los Angeles, CA) with a minimum detection limit of 0.1 ng/ml. The intraassay coefficients of variation were 3% (low reference), 3% (medium reference), and 4% (high reference). The interassay coefficients of variation were 7% (low reference, mean 1.69 ng/ml), 7% (medium reference, mean 2.62 ng/ml), and 1% (high reference, 11.66 ng/ml).

Plasma estradiol concentrations were determined using a commercial double-antibody radioimmunoassay kit (Catalog # KE2D5; Diagnostics Products Corporation) with a minimum detection limit of 1 pg/ml. Estradiol standards were made in charcoal-stripped bovine serum with a range of 1–200 pg/ml. The intraassay coefficients of variation for estradiol were 8% (low reference), 9% (medium reference), and 6% (high reference). The interassay coefficients of variation were 5% (low reference, mean 10.51 pg/ml), 6% (medium reference, mean 16.28 pg/ml), and 10% (high reference, mean 34.89 ng/ml). Estradiol data were analyzed only for the ovulatory wave to avoid confounding by estradiol 17ß treatment given for ovulation synchronization.

To estimate the level of stress due to frequent blood sampling, plasma cortisol concentrations were measured in frequent plasma samples (Days 8 and 18 of the IOI; 0-, 2-, 4-, 6-, 8-h samples) by competitive immunoassay (Catalog # LKCO5; Immulite; Diagnostic Products Corporation) [30]. Cortisol concentrations were compared with samples from other animals not used for frequent blood sampling (Days 8 and 18 of the IOI; 0- and 8-h plasma sample). The range of the standard curve was 10–500 ng/ ml, with minimum detection limit of 2 ng/ml. The intraassay coefficient of variation for cortisol was 7% (low reference, mean 48 ng/ml), 7% (medium reference, mean 122 ng/ml), and 6% (high reference, mean 363 ng/ ml).

Data Analysis

Dominant and first subordinate follicles of each wave were identified by retrospective analysis of ovarian sketches. The dominant follicle was defined as the largest follicle of the wave, first identified at 4–5 mm in diameter. The first subordinate follicle was defined as the second largest follicle originating from the same cohort of follicles [12, 31, 32]. The day of follicular wave emergence was defined as the day when the dominant follicle was first detected at 4–5 mm of diameter [12, 15, 31, 32]. The number of waves during the IOI was identified for each cow. The proportion of cows with two- or three-wave IOI and the proportion of mother-daughter pairs with the same or different wave patterns were analyzed by Fisher exact test. Interwave intervals (IWI), defined as the period between emergences of two successive waves, were calculated. Single-point numerical data (e.g., day of wave emergence, IOI, IWI, ovulatory diameter) were compared between old and young cows by Student t-test.

To characterize day-to-day changes in follicle numbers, follicles were categorized according to diameter (2–3 mm, 4–5 mm, and 6–8 mm). Data were centralized to wave emergence and analyzed by analysis of variance for repeated measures using the mixed procedure of the Statistical Analysis System (SAS; version 8.2 for MS Windows; SAS Institute Inc., NC) to determine the effects of age (old cows vs. daughters) and day of wave [31, 33]. Similarly, the diameters of the dominant and first subordinate follicles of each wave and gonadotropin data (FSH, LH) were centralized to wave emergence to determine the effects of age (old cows vs. daughters) and day of the IOI by analysis of variance for repeated measures. To determine temporal association between FSH peaks and wave emergence, FSH data were also analyzed by centralization to the peak of FSH (defined as highest value of FSH detected before wave emergence). Plasma LH concentrations in frequent blood samples were analyzed to compare LH-pulse frequency, pulse amplitude, mean and basal concentrations between groups (old cows vs. daughters) on Day 8 vs. Day 18 of the IOI (Day 0 = ovulation). Cortisol data were analyzed to determine the effect of age, day of IOI (Day 8 vs. Day 18), and sampling (frequently sampled animals vs. others not used for frequent plasma sampling).

Corpus luteum (CL) diameters and plasma progesterone concentrations were compared between groups from 0 to 15 days after the first ovulation of the IOI and from 0 to –5 days from the second ovulation by analysis of variance for repeated measures. Plasma estradiol values were centralized to the second ovulation (Day 0 to Day –7) and analyzed by analysis of variance for repeated measures to determine the effects of age during the development of the ovulatory follicle.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Interovulatory Interval and Wave Characteristics

The ovulation synchronization procedure resulted in tight synchrony among cows. Ovulation was detected between 48 and 72 hr after prostaglandin treatment in 19 of 20 cows (95%). The remaining cow ovulated 192 h after prostaglandin treatment and had a short interovulatory interval (9 days); therefore, data from this daughter were excluded from analyses. The proportion of cows with two follicular waves during the IOI was similar (P = 0.6) between old cows (6/10, 60%) and their daughters (7/9, 78%). The remainder had three waves of follicular development during the IOI. The difference in the proportion of mother-daughter pairs with the same vs. different wave patterns (6/ 9 vs. 3/9) was not significant (P = 0.35).

The duration of the IOI for two-wave and three-wave patterns was 20 and 23 days, respectively (P = 0.003). The mean days of wave emergence were 0.5 ± 0.1 and 10.5 ± 0.4 for two-wave IOI and 0.5 ± 0.2, 9.0 ± 0.5, and 15.83 ± 0.7 for three-wave IOI. For constructing day-to-day profiles (Figs. 1, 3, and 6), the mean days of wave emergence were rounded off to Days 0 and 10 for two-wave IOI and 0, 9, and 16 for three-wave IOI. The day of emergence of wave 1 was similar (P = 0.88) between two-wave (0.5 ± 0.1, n = 13) and three-wave IOI (0.5 ± 0.2, n = 6), but wave 1 emerged later (P = 0.04) in old cows than in their daughters (0.7 ± 0.2, n = 10 vs. 0.2 ± 0.2, n = 9, respectively; data combined from two- and three-wave IOIs). The remaining characteristics of follicular waves during the interovulatory interval in old cows and their daughters are summarized in Table 1. There was no difference between age groups in the IOI, IWI, or the day of wave emergence.



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  		FIG. 1. Number of follicles 2–3 mm (A), 4–5 mm (B), and 6–8 mm (C) in diameter (mean ± SEM) in old cows (n = 6) and their daughters (n = 7) in two-wave interovulatory intervals. Data for each wave were analyzed relative to the day of wave emergence (black triangles). *, Values between groups differ (P ≤ 0.05)



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  		FIG. 3. Diameter profiles (mean ± SEM) of the dominant (Dom) and first subordinate (Sub) follicles in old cows (n = 6 in A and n = 4 in B) and their daughters (n = 7 in A and n = 2 in B) in two-wave and three-wave interovulatory intervals (black triangles indicates wave emergence)



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  		FIG. 6. Plasma FSH (A) and LH (B) concentrations (mean ± SEM) in old cows (n = 6) and their daughters (n = 7) in two-wave interovulatory intervals. Data were analyzed relative to wave emergence (black triangles)


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  		TABLE 1. Comparison (mean ± SEM) between old cows and their daugh ters in follicular wave characteristics during one IOI

Follicle Numbers

Changes in the number of follicles in different size categories (2–3, 4–5, and 6–8 mm) are illustrated in Figure 1. No difference between age groups was detected in the number of 2- to 3-mm follicles per wave (Fig. 1A), but fewer (P = 0.01) 4- to 5-mm follicles were detected in wave 1 of old cows with two-wave IOI than in that of their daughters (Fig. 1B). For all follicular waves combined, fewer (P = 0.04) 4- to 5-mm follicles were detected during the period encompassing wave emergence in old cows compared with their daughters (old, n = 24 waves, vs. daughters, n = 20 waves; Fig. 2A). This was also reflected in a lower peak number of 6- to 8-mm follicles in old cows (specific day comparison P = 0.02; Fig. 1C).



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  		FIG. 2. A) Changes in the number of 4- to 5-mm follicles (mean + SEM) around the time of wave emergence (black triangle; all waves combined) in old cows (n = 24 waves) and their daughters (n = 20 waves). B) FSH concentrations (mean ± SEM) in old cows (n = 24 waves) and their daughters (n = 20 waves) relative to the FSH peak (Day 0). The FSH peak preceded wave emergence (black triangle) by 21 h in old cows and 17 h in their daughters (P = 0.4)

Follicle and Corpus Luteum Diameters

The diameter profiles of dominant and first subordinate follicles of anovulatory and ovulatory waves are illustrated in Figure 3. The diameter profile of the ovulatory follicle of old cows with two-wave IOI was smaller than that of their daughters (P = 0.04; Fig. 3) whereas no age-related effect was detected in cows with three-wave IOI (P = 0.83). When data from two- and three-wave IOI were combined, the mean diameter of the dominant follicle on the day before ovulation was smaller in old cows than in their daughters (12.3 ± 0.5, n = 10, vs. 13.9 ± 0.5, n = 9; P = 0.04). As expected, the diameter of the dominant follicle on the day before ovulation tended to be smaller in three-wave IOI than in two-wave IOI (11.9 ± 0.3, n = 6, vs. 13.5 ± 0.5, n = 13; P = 0.06). There was no effect of age on diameter profiles of the first subordinate follicles in either two-wave (P = 0.63) or three-wave IOI (P = 0.59). In old cows with two-wave IOI, the CL profile (Days 0– 15) tended to be smaller than in their daughters (P = 0.09; Fig. 4A), while no differences in CL diameter were detected for three-wave IOI (P = 0.69; Fig. 5A).



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  		FIG. 4. Profiles (mean ± SEM) of CL diameter (A) and plasma progesterone concentration (B) in two-wave interovulatory intervals of old cows (n = 6) and their daughters (n = 7). Data were analyzed from Day 0 (ovulation) to Day 15 and from Day –5 to Day 0 (subsequent ovulation)



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  		FIG. 5. Profiles (mean ± SEM) of CL diameter (A) and plasma progesterone concentration (B) in three-wave interovulatory intervals of old cows (n = 4) and their daughters (n = 2). Data were analyzed from Day 0 (ovulation) to Day 17 and from Day –5 to Day 0 (subsequent ovulation)

Gonadotropins and Cortisol

Characteristics of plasma concentration of gonadotropins in old cows (n = 10) and their daughters (n = 9) are summarized in Table 2. Mean FSH concentrations during the IOI (averaged over all days of IOI) were higher in old cows than in their daughters (Table 2; plasma samples n = 42/ animal/group, P = 0.01). Concentrations of FSH were significantly higher during the first wave (two-wave IOI) and during the ovulatory wave (two- and three-wave IOI) in old cows vs. their daughters (Fig. 6A). A combined analysis of all waves (old, n = 24 waves, vs. daughters, n = 20 waves; Day –2 to +3 from wave emergence) demonstrated higher circulating FSH concentrations in older cows (P = 0.009). When data were centralized to FSH peak (old, n = 24 waves, vs. daughters, n = 20 waves; Fig. 2B), day (P < 0.0001) and age effects (P = 0.008) were observed, but the period between the FSH peak and wave emergence was identical in both age groups. There were no differences between young and old cows in the preovulatory FSH peak concentration or time of its occurrence (Table 2).


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  		TABLE 2. Comparison (mean ± SEM) between old cows and their daughters in plasma concentrations of gonad otropins and sex steroids during one IOI

No differences between age groups were detected in mean plasma concentration of LH during the IOI or during the preovulatory LH surge (Table 2 and Fig. 6B). LH characteristics were studied in frequent plasma samples collected on Days 8 and 18 of IOI (Table 3). As expected, there was a higher number of LH pulses during the low progesterone phase (Day 18 of IOI) than during the midluteal phase (Day 8; P = 0.003). The mean number of LH pulses, pulse amplitude, means, and basal concentrations (Table 3) were not different between age groups.


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  		TABLE 3. Comparison (mean ± SEM) of plasma LH characteristics between old cows (n = 6) and their daughters (n = 6) in serial samples obtained every 15 min for 8 hours (n = 33) on Day 8 and 18 (Day 0 = ovulation)

Cortisol concentrations were not different between age groups (P = 0.23) and day of IOI (P = 0.43) for cows from which frequent blood samples were taken (Fig. 7, A and B). Cortisol concentrations were not different between cows from which frequent blood samples were taken or not (P = 0.61; Fig. 7, C and D).



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  		FIG. 7. Plasma cortisol concentrations (mean ± SEM) in old cows (n = 6) and their daughters (n = 6) during an 8-h sampling period on Day 8 (A) and Day 18 (B; Day 0 = ovulation) and a comparison of cortisol concentrations in plasma samples of cows that were frequently sampled and others not used for frequent plasma sampling (C, D)

Progesterone and Estradiol

Progesterone concentrations in cows with two-wave and three-wave patterns changed over days of IOI (P < 0.0001; Figs. 4B and 5B). A tendency for lower circulating progesterone (P = 0.09) was observed in old cows with three-wave IOI. When data were combined between two-wave and three-wave IOI, luteal phase progesterone concentrations (Days 8–15) tended to be lower in old cows than in their daughters (P = 0.10; Fig. 8A). Mean and peak plasma progesterone concentrations during the IOI were numerically lower in old cows, but differences were not statistically significant (P = 0.16 and 0.11, respectively; Table 2).



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  		FIG. 8. Plasma concentrations of progesterone during the luteal phase (A; Days 8–15) and estradiol during the preovulatory phase (B; Days –7 to 0) in old cows (n = 10) and their daughters (n = 9)

The profile of plasma concentrations of estradiol during the 7 days preceding ovulation was greater (P = 0.01) in old cows than in their daughters (Fig. 8B). There was a progressive increase in estradiol levels in both age groups (day effect, P < 0.0001). The preovulatory peak in estradiol concentrations and the time of its occurrence were not different between age groups.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Based on similarities in ovarian follicular dynamics and endocrine control in cattle and women [13, 15], the bovine model has been proposed for studying reproductive events in women [13]. This study was designed to characterize age-related changes in follicular, luteal, and endocrine function in cattle approaching reproductive senescence and to validate the existing bovine model for study of reproductive events in women approaching menopause. Based on the existing human literature, we hypothesized that reproductive aging in cattle would be associated with elevated circulating concentrations of gonadotropins, reduced concentrations of steroid hormones, and accelerated follicle recruitment during wave emergence.

Ovulation was synchronized using estradiol and progesterone for logistical purposes and to minimize the effects of environment and nutrition on follicular development and endocrinology over time. Follicular dynamics following estradiol and progesterone treatment have been well characterized [34, 35], and follicle numbers or diameters of the three largest follicles were similar to that of spontaneous waves. Furthermore, cows synchronized with a similar protocol had pregnancy rates comparable with untreated controls [23, 24]. Therefore, we expected the interovulatory cycles after ovulation synchronization in the present study to be similar to those occurring naturally. Cattle in the herd from which these cows were taken are selected for fertility, i.e., cows that fail to produce a calf are systematically culled. Hence, the old cows used in this study represent the most fertile of the herd and indeed had a calf in the spring preceding the experiment. Based on one report that 55% of the herd is infertile by 13 yr of age [1], old cows in the present study represent the top half of the herd in terms of fertility. By design, we used 13- to 14-yr-old cows and their 1- to 4-yr-old daughters that were born and maintained on the same farm throughout their life span. The design allowed us to minimize the effects of environmental and genetic variations and the complicating issues of specific reproductive pathology.

Similar to women in advanced reproductive age [4, 6– 8], we detected that mean plasma FSH concentrations during interovulatory interval as well as circulating FSH concentrations in follicular waves were consistently higher in old cows than in their daughters. A rise in circulating FSH concentrations was considered the first sign of reproductive aging in women [7], despite the fact that the women still had regular menstrual cycles. Results of the present study extend those of a previous study done in older cattle in which elevated circulating FSH concentrations were detected during Days 6–12 of the estrous cycle [22]. Data were not analyzed relative to follicular wave emergence in the later study, but it is noteworthy that elevated FSH concentration observed in old cows in the present study followed the expected pattern associated with wave emergence, i.e., each wave was preceded by a surge in circulating FSH (Fig. 2B). An age-related rise in urinary FSH-ß subunit was also reported in rhesus monkeys with irregular menstrual cycles. However, unlike women, an increase in FSH was not observed in middle-aged monkeys with regular menstrual cycles [36]. Elevated circulating concentrations of FSH were also observed in older mice [34] and rats [35].

The follicular wave pattern was maintained in the old cows and the majority of mother-daughter pairs had the same wave pattern (six pairs out of nine). The two-wave pattern occurred in 60% of the estrous cycles of old cows and the three-wave occurred in the remainder, similar to their daughters. This pattern is also consistent with that observed in previous studies in heifers [12, 37–41] and is consistent with the results of a recent study in normal young (mean, 28 yr) women [15]. The hereditary, nutritional, and environmental factors affecting wave patterns are not well understood, but in one study, cows fed high- and low-energy rations favored two- and three-wave patterns, respectively [42]. The repeatability of wave patterns, if any, is also not understood and no conclusive hereditary inferences could be drawn from the limited number of mother-daughter pairs in this study.

The length of interovulatory and interwave intervals did not change with age in the present study, similar to an earlier study in cattle where the length of estrous cycle did not differ among age groups [22]. A tendency for shorter menstrual cycles in older women was reported [4] and has been attributed to a short early follicular phase (interval from onset of menstruation to FSH rise) [4, 11]. Authors suggested that the early FSH rise in older women resulted in early dominant follicle selection [11]; however, others [15] have shown that the FSH rise is unrelated to the onset of menstruation and is associated with follicular wave recruitment rather than selection of the dominant follicle. The duration of the ovulatory wave (period from wave emergence to ovulation) was similar between old cows and their daughters in the present study. Similarly, the length of the late follicular phase in women (period from initial FSH rise to preovulatory gonadotropin surge) was not different between age groups [11].

The first wave of the estrous cycle in old cows emerged 12 h later than in their daughters, but there was no such age effect on emergence of other waves. It is interesting to note that, despite the short delay in emergence of the first wave, the interval between the preovulatory gonadotropin surge and ovulation at the end of our study period, and the interval between exogenous estradiol and induced ovulation at the beginning of the study were not affected by age. This appears contrary to a study in older women in which an early FSH peak was detected after recovery from hypothalamic-pituitary-gonadal axis suppression [11].

Fewer 4- to 5-mm follicles were recruited into a wave in old cows even though comparable numbers of 2- to 3-mm follicles were available at the time of wave emergence in young and old cows. This is contrary to our hypothesis that higher FSH concentrations in old cows would result in greater follicular recruitment. This hypothesis was also based on a presumed increased rate of follicle loss during menopause transition [5] as levels of FSH rise [4, 6–8] and on recent studies in cattle in which 1- to 3-mm follicles were found to be sensitive to FSH and develop in a wavelike pattern [31]. Based on our results, we speculate that increased FSH in old cows is able to stimulate small (2–3 mm) follicles from the dwindling ovarian follicular pool but may not be able to sustain their growth beyond the initial stages of wave emergence. In this study, we did not assess the rate of primordial, preantral, and early antral (<2 mm) follicle loss by atresia or their contributions to age-related follicle number decline. Reduced recruitment of 4- to 5-mm follicles into the follicular wave despite elevated circulating concentrations of FSH in old cows may be a result of 1) reduced numbers of granulosa cells in follicles, 2) reduced numbers of gonadotropin receptors per granulosa cell, 3) impaired receptor-hormone binding, 4) reduced responsiveness of granulosa cell after receptor-hormone binding, or 5) changes in the intrinsic ovarian follicle growth factor systems. Altered receptor-hormone interactions may also be involved in the decrease in superovulatory response reported in older women [43] and cattle [44]. In this regard, an age-related reduction in binding of FSH to its receptors was demonstrated in FSH-R heterozygous and wild-type mice [45]. FSH-R heterozygous mice show a rise in FSH levels with age, undergo accelerated follicle loss and reproductive aging, and have a shorter reproductive life than wild-type mice [46].

The ovulatory follicle of old cows with a two-wave pattern was smaller at the time of ovulation than that of young cows in the present study. The ovulatory follicle grew for the same number of days as in the daughters (no difference in duration of the ovulatory wave) but apparently at a slower rate (Fig. 3). This may also be due to reduced numbers or sensitivity of gonadotropin receptors in follicles of aging ovaries. A tendency for a smaller ovulatory diameter was observed in older women in some studies [4, 6] but not in others [11]. It is paradoxical that, although the diameter of the ovulatory follicle was smaller in old cows, ovulatory wave estradiol concentrations were higher in old cows than in their daughters. The latter is consistent with previous studies in which higher estradiol concentrations were reported during the preovulatory phase in older cattle [22] and during the follicular phase in older women [4].

Corpus luteum diameter tended to be smaller in old cows in the present study and may be associated with a smaller ovulatory follicle diameter. Luteal diameters correlated well with the observation that old cows in this study tended to have lower circulating concentrations of progesterone during the luteal phase, similar to the findings in an earlier study [22]. In women, progesterone concentrations also decrease in menopause transition [7]. The cause-and-effect relationship between low progesterone and subsequent age-related effects on follicular dynamics (i.e., smaller ovulatory follicle, fewer 4- to 5-mm follicles in the first wave, and delayed emergence of the first wave) remains to be elucidated.

There was no age effect on circulating LH concentrations or LH-pulse frequency, similar to previous studies in cattle [22] and women [2, 7]. In both age groups, LH-pulse frequency was higher during the low progesterone phase (Day 18) compared with the high progesterone phase (Day 8). The level of stress (assessed by circulating cortisol concentrations) was not different between age groups or between animals from which blood samples were taken intensively (every 15 min for 8 h) or less frequently (daily).

To summarize, an increased circulating concentration of FSH was detected in old cows but there was no change in LH. Old cows had higher circulating estradiol during preovulatory phase but progesterone concentrations tended to be lower during the luteal phase. Thus, our hypothesis that aging in cattle is associated with elevated gonadotropin and reduced steroid hormone concentrations was partially supported, and these changes were consistent with those reported during early reproductive aging in women. Reduced follicular recruitment (4–5 mm) at wave emergence was observed in old cows despite comparable numbers of 2- to 3-mm follicles between young and old, indicating possible diminished follicular responsiveness to FSH in older cattle. Thus, our hypothesis that reproductive aging will result in increased follicular recruitment was not supported. The ovulatory follicle grew more slowly and to a smaller maximum diameter in old vs. young cows. We conclude that changes in follicular dynamics and their endocrine control in 13- to 14-yr-old cows were similar to those previously reported in women approaching menopause and that the bovine model is suitable for the study of reproductive aging in women.

Observed differences in ovarian function between old and young cows in this study may be expected to be a conservative estimate of changes occurring during the transition to reproductive senescence and may be harbingers of age-related infertility. The bovine model may be particularly useful for addressing issues relevant to age-related infertility in women, such as 1) test of the hypothesis that antral follicle count is an accurate predictor of ovarian follicle reserve, 2) study of nuclear and cytoplasmic changes in the oocyte associated with subfertility and mechanisms associated with chromosomal aberrations, 3) identification of oocyte or granulosa cell markers of fertility, 4) development of interventional strategies for improving ovarian stimulation and oocyte competence, and 5) elucidation of the role of telomere length and telomerase activity in aging somatic and reproductive tissues.


   ACKNOWLEDGMENTS
 
The authors gratefully acknowledge the assistance of summer students Jasrajbir Batth, Laura Nauta, and Karen Kemp for data collection and Susan Cook for assistance with hormone assays. We also thank Bill Kerr and his staff at the Goodale Research Farm, University of Saskatchewan, for animal care and maintenance.


   FOOTNOTES
 
1 Supported by grants from the Natural Sciences and Engineering Research Council of Canada. Back

2 Correspondence. FAX: 306 966 7405; jaswant.singh{at}usask.ca Back

Received: 2 December 2004.

First decision: 19 January 2005.

Accepted: 28 February 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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