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Biol Reprod 2004, 10.1095/biolreprod.104.028548
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BIOLOGY OF REPRODUCTION 71, 804–812 (2004)
DOI: 10.1095/biolreprod.104.028548
© 2004 by the Society for the Study of Reproduction, Inc.

Neuroendocrinology

Regulatory Roles of Leptin at the Hypothalamic-Hypophyseal Axis Before and after Sexual Maturation in Cattle1

D.A. Zieba3,4,5, M. Amstalden3,4,5, S. Morton3,4,5, M.N. Maciel3,4,5, D.H. Keisler6, and G.L. Williams2,3,4,5

Animal Reproduction Laboratory,3 Texas A&M University Agricultural Research Station, Beeville, Texas 78102 Department of Animal Science4 Center for Animal Biotechnology and Genomics,5 Texas A&M University, College Station, Texas 77843 Department of Animal Science,6 University of Missouri, Columbia, Missouri 65211


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Studies assessed, either directly or indirectly, the role of GnRH in leptin-mediated stimulation of LH release in cattle before and after sexual maturation. In experiment 1, the objectives were to determine whether leptin could acutely accelerate the frequency of LH pulses, and putatively GnRH pulses, in prepubertal heifers at different stages of development. In experiment 2, we determined directly whether acute, leptin-mediated increases in LH secretion in the fasted, mature female are accompanied by an increase in GnRH secretion. Ten-month-old prepubertal heifers (experiment 1) fed normal- (n = 5) and restricted-growth (n = 5) diets received three injections of saline or recombinant ovine leptin (oleptin; 0.2 µg/kg body weight, i.v.) at hourly intervals during 5-h experiments conducted every 5 wk until all normal-growth heifers were pubertal. Leptin increased mean concentrations of circulating LH regardless of diet, but pulse characteristics were not altered at any age. In experiment 2, ovariectomized, estradiol-implanted cows (n = 5) were fasted twice for 72 h and treated with either saline or oleptin i.v. (as in experiment 1) on Day 3 of each fast. Leptin increased plasma concentrations of LH and third ventricle cerebrospinal fluid concentrations of GnRH, and increased the amplitude of LH and the size of GnRH pulses, respectively, on Day 3 of fasting compared to saline. Overall, results indicate that leptin is unable to accelerate the pulse generator in heifers at any developmental stage. However, leptin-mediated augmentation of LH concentrations and pulse amplitude in the nutritionally stressed, mature female are associated with modifications in GnRH secretory dynamics.

gonadotropin-releasing hormone, leptin, luteinizing hormone, puberty


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Among the neuroendocrine actions of the adipocyte-derived peptide, leptin, its stimulatory effects on the hypothalamic-hypophyseal-gonadal axis are of particular significance. Leptin clearly plays an important role in signaling nutritional status to the central reproductive axis [1, 2], and appears to play at least a permissive role in the initiation of puberty [3–5].

In fasted cows, leptin-mediated changes have included an increase in baseline and overall mean concentrations of LH, and an augmentation of the size of individual pulses of LH [6, 7]. These findings, coupled with more recent observations using perifused anterior pituitary explants, are consistent with the view that the effects of leptin on LH secretion in the sexually mature ruminant female may reside predominantly at the adenohypophyseal level [8]. However, conclusions regarding leptin's inability to affect hypothalamic GnRH secretion in fasted cattle were not made from direct measurements of GnRH in vivo. Moreover, our recent demonstration that exogenous leptin can prevent fasting-mediated reductions in the frequency of pulses of LH in intact, sexually immature heifers [9] implies a direct action at the hypothalamic level in this model. Importantly, expression of leptin receptor (LR) mRNA has been demonstrated in both the anterior pituitary gland and hypothalamus [10], and the LR has been identified within regions rich in GnRH neurons such as the arcuate nucleus, the medial preoptic area, and the median eminence [11, 12].

Experiments reported herein examined two hypotheses regarding potential pathways through which leptin might modulate LH secretion in cattle before and after sexual maturation. In experiment 1, we sought to test the hypothesis that leptin is capable of acutely accelerating the frequency of LH pulses at a critical point of development in normal-fed, or growth-restricted (or both), prepubertal heifers. The ability to create such effects in one or both groups would confirm a role for leptin in initiating puberty and reinforce the idea that, in this animal model, the major effects of leptin are at the hypothalamic level. In experiment 2, we tested the hypothesis that the ability of leptin to increase circulating concentrations of LH in the sexually mature female, effects that to date have been accounted for primarily within the adenohypophysis [6–8], may also be attributed in part to direct effects on hypothalamic GnRH secretion.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All animal-related procedures used in these studies were approved by the Institutional Agricultural Animal Care and Use Committee of The Texas A&M University System.

Experiment 1: Acute Effect of Leptin on Pulsatile Secretion of LH in Prepubertal Heifers Subjected to Normal and Restricted Growth Diets

Neuroendocrine model The neuroendocrine axis of the prepubertal female, including the heifer, is characterized by infrequent, high-amplitude pulses of LH [13, 14]. As the female matures, the frequency of pulses of LH increases until the pubertal ovulation. Based on evidence in male ruminants [15, 16], female monkeys [17], and female rats [18], the increase in LH secretion at puberty is accompanied by concomitant increases in the frequency, amplitude, or both of GnRH pulses within the hypothalamic neurosecretory network and hypophyseal portal vessels. Therefore, determining GnRH pulse-generator activity indirectly by estimating the frequency of LH pulses is a well-documented and widely accepted approach in experimental animal models that are characterized by marked changes in pulse frequency [19, 20]. The physiological model examined in experiment 1 met this criterion, and allowed us to avoid the use of invasive surgical techniques for assessing GnRH secretion directly. Thus, by estimating the frequency and amplitude of pulses of LH, we sought to evaluate indirectly whether the GnRH pulse generator changes its sensitivity to leptin as puberty approaches. Should this occur, we expected acute treatments with leptin to be capable of accelerating the frequency of LH pulses at a critical period or periods before the onset of the first pubertal ovulation.

Procedures Experiments were initiated on December 17, 2002 with 10 spring-born, prepubertal (Brahman x Hereford) heifers with a starting body weight (BW) of 274.9 ± 5 kg. At the beginning of the experiment, heifers were 10 to 10.5 mo of age. Based on previous experience with heifers of this breed type [9], we expected heifers fed normal growing diets to have their first pubertal ovulation between 14 and 15 mo of age. Each heifer was assigned randomly to one of two dietary groups: 1) normal growth (diets formulated with Coastal bermudagrass hay and grain to promote a weight gain of approximately 0.68 kg/day), and 2) restricted growth (heifers were limit-fed average-quality Coastal bermudagrass hay only to promote a weight gain of approximately 0.3 kg/day). Heifers were maintained in pens (25 x 9 m) and serum concentrations of progesterone were determined in twice-weekly blood samples collected by caudal venipuncture to monitor pubertal status.

The experimental design for leptin challenge was a switchback design in which each animal served as her own control. Every 5 wk, beginning at the onset of dietary treatments, heifers received on each day of a 2-day experiment either saline or leptin treatment. The initial saline-leptin treatment sequence (Week 1) for each heifer was assigned randomly, and then alternated thereafter. Highly purified recombinant oleptin was provided by Dr. Arieh Gertler (Hebrew University of Jerusalem, Rehovot, Israel) as reported previously [21]. The dose of leptin chosen was based on a previous study in our laboratory in which oleptin demonstrated an inverse, dose-dependent influence on basal secretion of LH in fasted cows [7].

Treatments were administered i.v. and consisted of the following: 1) physiological saline (control) and 2) recombinant ovine leptin (oleptin; 0.2 µg/kg BW) [7], three times at 0, 1, and 2 h of each 5-h experiment. On the day before the start of each experiment (Day –1), heifers were fitted with jugular catheters (polyethylene tubing, 1.4 mm inside diameter [i.d.] x 1.9 mm outside diameter [o.d.]; Becton Dickinson, Parsippany, NJ) for intensive blood sampling. On each experimental day, heifers were placed in indoor stanchions, and blood samples were collected at 10-min intervals for 5 h, with the first sample collected immediately before the first injection at time 0. Blood samples were dispensed into tubes containing a solution of 150 µl heparin (10 000 UI/ml) and 5% EDTA, and placed immediately on ice. During intensive blood sampling, the volume of blood collected (10 ml) was replaced with an equal volume of saline or heparinized (300 IU/ml) saline during catheter flushing. Serum (nonintensive, caudal blood samples) or plasma was obtained by centrifugation and stored at –20°C until hormone assays were conducted. The experiment ended on May 30, 2003 when all heifers from the normal-growth group had reached puberty. Heifers were considered pubertal when concentrations of progesterone were ≥1 ng/ml in two or more consecutive weekly samples. Therefore, the experiment encompassed 25 wk and a total of five experimental periods for restricted-growth heifers (n = 5). As normal-growth heifers reached puberty, they were removed from the treatment and sampling schedule. Therefore, the number of normal-growth heifers included in experimental periods one through five was 5, 5, 5, 4, and 2, respectively.

Experiment 2: Acute Effect of Leptin on GnRH and LH Secretion in Mature, Ovariectomized Cows During Nutritional Stress

Neuroendocrine model In previous experiments reported from this laboratory, we demonstrated the ability of recombinant oleptin to increase the secretion of LH in the sexually mature, ovariectomized cow fasted for 56 to 72 h [6–8]. In this animal model, while fasting hypersensitizes the central reproductive axis to leptin, it does not cause a suppression of the frequency of LH pulses [6–8]. As a result, the ability of leptin to stimulate LH secretion was characterized by changes that appeared to be accounted for primarily by effects at the adenohypophyseal level [8]. Nonetheless, our experiments were unable to completely eliminate the possibility of hypothalamic effects of leptin that would result in increased secretion of GnRH. Therefore, for experiment 2, we measured GnRH secretion directly in third ventricle cerebrospinal fluid (CSF) as described previously in detail from our laboratory [22]. This approach allows the measurement of hypothalamic pulse-generator activity, with individual pulses of GnRH highly correlated with both portal vessel pulses of GnRH [23] and peripheral vascular pulses of LH [24]. These relationships have been confirmed in many species, including monkeys [24], rabbits [25], and sheep [23]. In addition, similar to our previous leptin-related experiments in mature cows, we used the ovariectomized, estradiol-implanted female to test the effects of leptin. This neuroendocrine model has been used extensively by our laboratory and others to study nutrition-reproduction interactions in cattle, avoiding confounding effects of ovarian cyclicity [26, 27].

Procedures Five mature, ovariectomized cross-bred beef cows were used, each bearing an s.c. estradiol implant constructed from Silastic tubing (3.35 mm i.d. x 5.65 mm o.d. x 70 mm; Dow Corning, Konigsberg Instruments, Inc., Pasadena, CA) to maintain circulating concentrations of estradiol at 2–4 pg/ml [22]. Cows were in moderate to good body condition (BC = 4–5; 1–9 scale) and were fed once daily at 0700 h a diet formulated to provide 100% of the National Research Council [28] recommendations for maintenance before the beginning of the experiment. Cows were surgically fitted with third ventricle cannulas as described previously [6, 22]. The location and function of cannulas were verified by radiography and continuous flow of CSF. A period of at least 3 wk was allowed for cows to recover from surgery.

Animals were placed frequently in stanchions to familiarize them with the experimental conditions and minimize the effects of stress on pulsatile secretion of GnRH [29]. During the experiment, all cows were fasted for 72 h with free access to water. One day before the start of fasting (Day –1), cows were fitted with jugular catheters for intensive blood sampling. At the same time, animals were treated prophylactically with antibiotics as reported previously [22]. Approximately 2 h later, 250 mm of polyethylene tubing (0.58 mm i.d. x 0.96 mm o.d.; Intramedic Clay Adams Brand, Becton Dickinson, Sparks, MD) was inserted using aseptic technique through the third ventricle to guide the cannula so that the distal end projected 5–6 mm past the end of the cannula and into the ventricle. The proximal end of the tubing extended approximately 50–60 mm above the tip of the guide cannula. Tubing was adjusted until CSF flowed easily using a blunt, 22-gauge needle and tuberculin syringe. The tubing was then plugged until later use.

On the day of the experiment, cows were placed in stanchions, CSF flow was confirmed, and the end of the tubing was connected to another 750-mm section of polyethylene tubing. The collection end of the tubing was secured about 50–60 cm away from the cow's head to facilitate semiremote sampling. Blood samples (10 ml), via extensions connected to the jugular catheter and remotely secured, were collected simultaneously with CSF samples (600 µl) at 10-min intervals for 5 h on Days 0 and 3 of each fast. In all cases, void volumes of CSF and blood created by the indwelling tubing and their extensions were discarded before sample collection. On Day 3, treatments (saline or oleptin) were injected i.v. three times at 0, 1, and 2 h, with the first sample was collected immediately before the first injection at time 0. Treatments consisted of the following: 1) physiological saline (control), or 2) oleptin (0.2 µg/kg BW) [7] in a switchback design such that each cow received one of each of the treatments in random order approximately 4 wk apart.

Blood samples were dispensed into tubes containing 150 µl of a solution containing heparin (10 000 IU/ml) and 5% EDTA, and placed on ice immediately. Plasma was separated by centrifugation and stored at –20°C until LH analysis. CSF samples were placed on ice immediately and, within 30 min, stored at –70° C until assayed for GnRH.

Radioimmunoassays

To confirm the pubertal status of heifers in experiment 1, serum concentrations of progesterone were assayed in twice-weekly samples collected beginning 4 wk before and continuing throughout the study using the Coat-A-Count assay kit (Diagnostics Product Corporation, Los Angeles, CA) as reported previously [30]. Circulating concentrations of leptin were determined in samples collected twice weekly from a subset of six heifers (three normal growth and three restricted growth) and during both experimental days of experimental periods 1 (10.5 mo of age), 3 (13 mo of age), and 5 (15.5 mo of age). The latter included samples collected every 30 min for the first 3 h and every 60 min for the remaining 2 h using a highly specific oleptin RIA validated for use in bovine serum [31]. Use of this assay for determining plasma concentrations of leptin in the bovine has been reported previously by our laboratory [8, 9, 27]. For experiment 2, plasma leptin was assayed on Days 0 and 3 of each fast in all cows every 30 min for the first 3 h, and every 60 min for the remaining 2 h on each day. Plasma concentrations of LH were determined with a validated RIA [32] in samples collected at 10-min intervals for 5 h on each experimental day in both experiments 1 and 2. Measurement of GnRH in CSF collected at 10-min intervals for 5 h on Days 0 and 3 in experiment 2 was performed in duplicate as described previously [22], except that assay volumes ranged from 150 to 250 µl, and antiserum BDS 037 (kindly provided by Dr. Alain Caraty, Physiologie de la Reproduction des Mammifères Domestiques, INRA, Nouzilly, France) was used as the source of primary antibody. Final working dilution of the antiserum was 1:150 000. Intraassay and interassay coefficients of variation (CV) for the LH, progesterone, and GnRH assays were 6% and 8%, 11% and 8%, and 11% and 6.5%, respectively. The intraassay CV of the leptin assay was 3.1%.

Statistical Analysis

In experiments 1 and 2, the frequency and amplitude of LH and GnRH pulses were determined with the aid of a pulse-detection algorithm, Pulsefit 1.2 [33], with areas under each pulse determined using the trapezoid rule. Temporal coincidence between GnRH and LH pulses within cows was as defined by Gazal et al. [22] using the methodology of Woller et al. [34]. Frequency, amplitude, and size (area under the curve; AUC) of LH and GnRH pulses, plasma or serum concentrations of leptin and LH (experiments 1 and 2), and CSF concentrations of GnRH (experiment 2) were analyzed by the general linear models (GLM) procedure (PROC GLM) of the Statistical Analysis System (SAS) [35]. Sources of variation for analysis of variance in experiment 1 included treatment, diet, experimental day, and period within experimental day. Heifer within treatment x diet served as the error term to test for main effects of treatment for each experimental day. For experiment 2, sources of variation for the initial model included treatment, day, and treatment x day. However, because differences in hormone concentrations existed on Day 0, data were examined in two ways: transformation to a percentage of Day 0 and covariate analyses using mean concentration on Day 0 as the covariate to test main effects of treatment on Day 3. The Proc CORR procedure of SAS (SAS Institute, Cary, NC) was used to perform Pearson simple correlations between pulses of GnRH and LH.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1

Serum concentrations of progesterone remained below 0.3 ng/ml throughout the study in all heifers within the restricted-growth group, indicative of a continuance of the prepubertal state. In the normal-growth group, serum concentrations of progesterone in each heifer also remained below 0.3 ng/ml until they individually exhibited their first pubertal ovulation between 13.5 and 15.5 mo of age. Daily body weight gains in the normal- and restricted-growth groups averaged 0.8 ± 0.09 and 0.3 ± 0.04 kg/day, respectively.

Mean plasma concentrations of leptin measured in a subgroup of six heifers over the entire study are shown in Figure 1A. Circulating leptin was about 70% lower (P < 0.001) in restricted-growth heifers compared to normal-growth heifers (Fig. 1A) throughout the study. Acute responses to injections of saline or leptin in both dietary groups averaged over experimental periods 1, 3, and 5 are presented in Figure 1, B and C. Injections of recombinant oleptin increased acutely (P < 0.002) the mean plasma concentrations of leptin in both dietary groups compared to saline treatment during each 5-h experiment. Plasma leptin was elevated about 1.7-fold in normal-growth and by more than 2-fold in restricted-growth heifers.



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  		FIG. 1. Mean concentrations of circulating leptin in prepubertal heifers in experiment 1. A) Serum concentrations of leptin in twice-weekly samples collected throughout the study, with mean concentrations of leptin lower (P < 0.001) in restricted-growth heifers compared to normal-growth heifers. Intravenous injections of saline (S) or oleptin (L; 0.2 µg/kg) were administered at 0, 1, and 2 h (B and C) and are represented by overall means for experimental periods 1, 3, and 5. Leptin increased (P < 0.002) mean (± SEM) plasma concentrations of leptin in both dietary groups compared to saline

Restricted-growth diets slightly but significantly lowered (P < 0.001) mean concentrations of LH compared to normal-growth diets beginning at experimental period 3 and continuing through the fifth and final period (0.66 ± 0.01 vs. 0.76 ± 0.01 ng/ml). This difference was likely the result of the lower amplitude (P < 0.001) of individual pulses of LH observed in the restricted-growth group compared to the normal-growth group (0.78 ± 0.1 vs 1.45 ± 0.2 ng/ ml), as both frequency of pulses and areas under the curve (AUCs) were not consistently lower in the restricted-growth group. Conversely, mean circulating concentrations of LH were slightly but significantly greater (P < 0.02) over all leptin treatment periods (0.73 ± 0.01 ng/ml) than during saline treatment periods (0.69 ± 0.01 ng/ml). However, this was not accounted for by detectable changes in frequency, amplitude, or size (AUC) of pulses in response to leptin injections at any age. Individual pulse patterns in representative heifers from the normal- and restricted-growth groups at three developmental ages (10.5, 13, and 15.5 mo) are presented in Figures 2 and 3.



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  		FIG. 2. Pulsatile pattern of LH release in a representative heifer (#204) from the normal-growth group treated with saline (S; left panels) or recombinant oleptin (L; right panels) at three developmental ages: 10.5, 13, and 15.5 mo. Pulses detected by Pulsefit [32] are denoted by an asterisk and are enumerated in parentheses



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  		FIG. 3. Pulsatile pattern of LH release in a representative heifer (#2645) from the restricted-growth group treated with saline (S; left panels) or recombinant oleptin (L; right panels) at three developmental ages: 10.5, 13, and 15.5 mo. Pulses detected by Pulsefit [32] are denoted by an asterisk and are enumerated in parentheses

Experiment 2

Mean concentrations of leptin on Days 0 and 3 of fasting are presented in Figure 4, A and B. Intravenous injections of oleptin on Day 3 increased (P < 0.001) mean concentrations of circulating leptin compared to saline (Fig. 4B). Patterns of CSF GnRH and plasma LH secretion in two representative ovariectomized cows are shown in Figure 5. Over the 10 5-h periods of sampling that encompassed Days 0 and 3 of fasting, a total of 98 LH and 95 GnRH pulses were detected, with a correlation coefficient (R) of 0.90 (P < 0.001). Leptin treatment on Day 3 of fasting increased mean concentrations of LH compared to saline (2.84 ± 0.06 vs. 2.37 ± 0.6 ng/ml; P < 0.001; Fig. 6A), which could be accounted for mainly by a 1.9-fold increase (P < 0.04) in the amplitude of LH pulses (2.22 ± 0.4 vs. 1.21 ± 0.4 ng/ml; Fig. 6C). Neither frequency (Fig. 6B) nor size (AUC) of pulses (Fig. 6D) of LH were affected by leptin treatment. Coincident with leptin-mediated increases in mean concentrations of LH (Fig. 6A) and amplitude of pulses (Fig. 6C) was an increase (P < 0.058) in mean concentrations of CSF GnRH (6.52 ± 1.3 vs. 3.23 ± 1.3; Fig. 6A). This increase was accompanied by an increase (P < 0.03) in the mean size (AUC) of GnRH pulses after leptin compared to saline (Fig. 6D). The amplitudes of individual pulses of GnRH were characterized by large variability (Fig. 6C), and numerical differences were not statistically relevant.



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  		FIG. 4. Mean concentrations (± SEM) of leptin in saline and leptin-treated, ovariectomized, estradiol-replaced cows on Days 0 (A) and 3 (B) of fasting. Intravenous injections of oleptin (L) on Day 3 increased (P < 0.001) mean concentrations of circulating leptin compared to saline (S)



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  		FIG. 5. Pulsatile patterns of LH and GnRH release in two representative, ovariectomized, estradiol-replaced cows after saline or recombinant oleptin injections on Day 3 of fasting. Pulses detected by Pulsefit [33] are denoted by an asterisk and are enumerated in parentheses. Three subjectively identified peaks of GnRH are denoted by a plus sign (+). These peaks were not statistically significant, but preceded pulses of LH. Arrows indicate time of treatment with saline or leptin



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  		FIG. 6. Characteristics of GnRH (left panels) and LH (right panels) pulses in ovariectomized, estradiol-replaced cows on Day 3 of fasting after infusion of saline or recombinant oleptin. An increase in mean concentrations of CSF GnRH (*P < 0.09) and plasma LH (***P < 0.001) was observed during leptin-injection periods compared to saline-injection periods (A). In addition, the amplitude of LH pulses (**P < 0.06; C) and mean size (AUC) of GnRH pulses (**P < 0.03; D) were greater during leptin-injection periods compared to saline-injection periods. Neither LH nor GnRH pulses frequency was affected by leptin (B)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In experiment 1, objectives were to test the ability of recombinant oleptin to stimulate an acute increase in the frequency of LH, and putatively GnRH, pulses at different stages of development in prepubertal heifers fed normal- or restricted-growth diets. Circulating leptin concentrations, as measured in a subgroup of heifers, were markedly reduced in restricted growth compared to the normal-growth heifers, a phenomenon that has been documented previously in animals subjected to chronic dietary energy restriction [36, 37]. Furthermore, normal- and restricted-growth heifers injected with recombinant oleptin exhibited increases in plasma leptin concentrations of 1.7-fold (normal growth) to more than 2-fold (restricted growth) during each of three 5-h experimental periods in which concentrations of leptin were examined. In a previous study [38], we have shown that chronic treatment with high levels of recombinant oleptin for up to 40 days is incapable of accelerating the timing of puberty in normally fed, prepubertal heifers. Based on those findings, we speculated that chronically elevated, high levels of leptin in the circulation may have actually induced a state of leptin resistance [39, 40], thus impeding sexual maturation. Therefore, in the current study we hypothesized that, if leptin serves as a maturational trigger for puberty, the central reproductive axis of normal- or restricted-growth (or both) heifers should become hypersensitized and respond acutely to modest, short-term increases in circulating leptin as the chronological time of expected puberty approached. Although acute treatments with leptin slightly increased mean concentrations of LH over all periods combined, an effect that could be of adenohypophyseal origin [6–8], leptin did not accelerate pulse frequency during any experimental period in either dietary group. Therefore, neither chronological age nor modest dietary energy restriction, adequate to delay puberty, appear to provide the requisite conditions for leptin-mediated increases in pulse frequency before puberty. Such results are in partial agreement with other recent reports in which the role of leptin in pubertal development of mice was reassessed [3]. In that study, it was noted that, while leptin reversed the delay in pubertal maturation secondary to food restriction, it did not advance the onset of puberty in normally fed mice.

The delay in puberty in restricted- compared to normal-growth heifers was as expected and is in agreement with a previous report [41]. Moreover, our observation that mean concentrations of plasma LH were lower over all periods in restricted-growth heifers compared to normal-growth heifers was also consistent with our expectations and observations of others [26]. This decline was associated with a consistent reduction in amplitude of individual pulses of LH. Our inability to detect long-term changes in pulse frequency due to dietary energy restriction was not unexpected, and is likely related to the intervals between our intensive sampling periods. During pubertal development in heifers, a measurable increase in the frequency of LH pulses from the prepubertal, low-frequency state is detected only during the final 50 days of maturation, and 24-h pulse patterns are required to reliably detect such transient changes [41].

Age at onset of puberty in heifers is regulated by genetics, dietary energy intake, growth rate, and adiposity [41, 42]. The neuroendocrine milieu at pubertal onset in male ruminants is characterized by frequent, high-amplitude pulses of LH [13, 14] that are accompanied concomitantly by pulses of GnRH in the hypophyseal portal circulation [15, 16]. However, to our knowledge, there have been no studies reporting the direct measurement of GnRH pulse patterns in the female ruminant during pubertal development. Instead, significant changes in the frequency of LH pulses during different physiological states of female cattle and sheep have been used to indirectly characterize GnRH pulsatility. Dietary energy restriction and restricted growth delay puberty primarily by preventing the development of high-frequency pulses of LH, which is due in part to heightened negative feedback sensitivity to estradiol [14, 41], and putatively, to lack of an adequate GnRH signal [15, 41, 42].

I'Anson et al. [43] reported that frequency and amplitude of GnRH pulses are altered by nutrition. In a group of growth-restricted female lambs, significantly fewer pulses of GnRH were detected compared to normally growing animals. Moreover, low-amplitude pulses of GnRH failed to induce coincident pulses of LH. The potential interaction of leptin within this complex system is unknown, and the minimal level of circulating leptin required to allow puberty to proceed normally has not been determined. In some studies, circulating leptin concentrations were reported to be stable throughout pubertal development [3, 44], whereas two other studies have reported a rise from prepuberty to postpuberty [45, 46]. In heifers, there is a linear increase in circulating leptin beginning at least 16 wk before puberty [47], and in human females, declines in leptin-binding activity in serum accompany pubertal increases in circulating leptin [48]. No changes in leptin binding activity were noted during pubertal development in heifers [47]. Collectively, the consensus of recently published studies and emerging concepts in this area of investigation indicate that leptin is probably not a trigger for puberty in normal females, but instead serves as a permissive signal or gate to the onset of puberty [3, 4].

Although evidence to support a role for leptin as a trigger for pubertal transition in heifers was not evident in experiment 1, other effects of leptin in cattle are clearly distinguishable. For example, we reported recently the ability of recombinant oleptin to prevent fasting-mediated reductions in the frequency of LH pulses in peripubertal heifers and to enhance responsiveness of the anterior pituitary to GnRH [9]. Therefore, in this animal model, it appears that the highly active, yet immature, pulse generator is exquisitely sensitive to dietary energy restriction, and these effects can be obviated by acute treatment with leptin. To the contrary, in older, sexually mature females with significantly more adipose reserves, while failing to suppress the frequency of pulses of LH [6–8], fasting appears to sensitize the animal to leptin to a large extent through direct effects at the adenohypophyseal level [8]. In this model, leptin-mediated increases in basal secretion of LH are not dependent on, and do not occur, in association with changes in pulse frequency [6–8]. Nonetheless, the designs of our previous experiments have not allowed us to completely rule out hypothalamic effects. Thus, experiment 2 was conducted to test the hypothesis that part of leptin's actions in the mature cow could be mediated through effects on hypothalamic GnRH secretion, an effect that could also plausibly account for the increase in mean circulating LH observed in prepubertal heifers (experiment 1). As expected, short-term fasting sensitized the hypothalamic-pituitary axis to leptin, and leptin treatments increased mean concentrations of LH in fasted but not in control-fed cows, similar to our previous reports [6–8]. These findings are also in agreement with recent studies in other species in which the effects of leptin on endogenous gonadotropin secretion occurred only in fasted rats [49], monkeys [50], or sheep [51], or following long-term food restriction in sheep [36]. Increases in mean LH in the current study were related to a measurable increase in the amplitude of LH pulses after leptin injections. Importantly, a concomitant rise in mean concentrations of CSF GnRH was also observed in fasted cows after i.v. infusion of oleptin but not in saline-treated animals. This rise was associated with an increase in the mean size (AUC) of GnRH pulses.

The relatively close temporal association between CSF GnRH and plasma LH in ovariectomized cows in this study supports our previous observations [22] and reports in the ewe [23] that indicate that pulses of LH are accompanied by similar patterns of GnRH release in CSF. However, results of the current study were not without inconsistency relative to specific changes in GnRH and LH pulse characteristics (i.e., LH: increased pulse amplitude; GnRH: increased pulse size). Currently, we have no explanation for these discrepancies, but they possibly relate to differences in our ability to detect and characterize the dynamics of hormone secretion and metabolism in separate fluid compartments (CSF vs. peripheral blood plasma). It has been noted that CSF GnRH pulses are of longer duration than portal GnRH pulses, and CSF GnRH pulses often peak after peripheral LH pulses [22, 23]. In ewes, a weak correlation between the actual amplitude of CSF GnRH pulses and jugular pulses of LH was observed, with a strong correlation between amplitudes of portal GnRH and jugular pulses of LH. Moreover, it was noted that portal GnRH pulses tended to have greater amplitude compared with CSF GnRH pulses [23]. I'Anson et al. [43] observed that high-amplitude pulses of GnRH in portal blood coincided with pulses of LH in growth-restricted, hypogonadotropic female sheep; however, small and low-amplitude pulses of GnRH were not accompanied by concomitant pulses of LH. Similarly, in heifers [52], the amplitudes of CSF GnRH pulses occurring without coincident LH pulses were significantly smaller than the amplitudes of GnRH pulses occurring coincident with pulses of LH.

Neuroendocrine mechanisms through which leptin could influence GnRH neuronal activity are still uncertain. The leptin receptor is present at both hypothalamic and adenohypophyseal loci [10, 53–55], and in vitro studies using explants collected from normal-fed rodents indicate that leptin can act directly at both sites [56, 57] to stimulate the release of GnRH and LH, respectively. However, much of leptin's influence on GnRH secretion may be through interneuronal signaling, because few GnRH neurons, if any, have been found to express the leptin receptor protein in the rat [58] and monkey [50]. Data from Watanobe [59] strongly suggest that leptin can act at both cell bodies and axon terminals of GnRH neurons to stimulate the release of the neurohormone in vivo, with higher sensitivity of the median eminence-arcuate nucleus to leptin in fasted than in fed rats. Based on increases in both receptor mRNA and protein levels [60, 61], fasting may enhance leptin receptor concentration in the arcuate nucleus. Similarly, expression of the full-length leptin receptor in the ventromedial hypothalamus was found to be much greater in feed-restricted ewes than in ewes that were well fed [53]. Also, negative energy balance induced by fasting has presynaptic actions that are conveyed by a reduction in excitatory GABAergic drive onto GnRH neurons. Treatment with exogenous leptin prevented this reduction, indicating that leptin can act presynaptically to restore afferent GABAergic drive to GnRH neurons in fasted animals [62].

In conclusion, current and previous work support the idea that leptin alone is not sufficient to initiate sexual maturation in prepubertal heifers. Moreover, leptin directly modulates hypothalamic GnRH secretion in mature, nutritionally stressed cattle, in addition to its clear and direct effects at the adenohypophysis. Therefore, leptin may act at both hypothalamic and adenohypophyseal centers to regulate gonadotropin secretion in cattle, and observable manifestations of these effects are dependent on the interaction of nutritional status with stage of sexual maturation.


   ACKNOWLEDGMENTS
 
We acknowledge the National Pituitary Hormone Program for providing LH preparations, Dr. Jerry Reeves for the LH antisera, Dr. Alain Caraty for GnRH antisera, and Dr. Arieh Gertler for ovine recombinant leptin. We also acknowledge with gratitude the careful animal care and assistance of Melvin Davis, Randle Franke, and Justin Kinnamon, and the technical assistance of Pat Chen.


   FOOTNOTES
 
1 Supported by USDA-NRI 00-35203-9132. Back

2 Correspondence: G.L. Williams, Animal Reproduction Laboratory, Texas A&M University Agricultural Research Station, 3507 Hwy 59 E, Beeville, TX 78102-9410. FAX: 361 358 4930; glwilliams{at}tamu.edu Back

Received: 18 February 2004.

First decision: 4 March 2004.

Accepted: 3 May 2004.


   REFERENCES
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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