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Biology of Reproduction 59, 1124-1130 (1998)
©Copyright 1998 Society for the Study of Reproduction, Inc.

Excitatory Amino Acid Regulation of Gonadotropin Secretion in Prepubertal Heifer Calves1

Ali Honaramooza, Ramesh K. Chandolia3,a, Andrew P. Bearda, , and Norman C. Rawlings2,a

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mechanisms controlling the pulsatile release of gonadotropins in prepubertal heifers are not completely understood. We examined the role of excitatory amino acid neurotransmitters, via activation of N-methyl-D-aspartate (NMDA) receptors, in the control of pulsatile LH and FSH release during prepubertal development in heifers. Hereford heifer calves received 4.7 mg/kg of N-methyl-D,L-aspartic acid (NMA), a potent NMDA receptor agonist (n = 5, i.v.), or saline (n = 5, i.v.), as single doses, at 4, 8, 12, 24, 36, and 48 wk of age. Blood samples were collected every 15 min, for 1 h before and 9 h after injection, on the days of treatment. Injection of NMA resulted in an acute release of LH (p < 0.001) in 0, 3, 3, 4, 5, and 5 calves (p < 0.01) and of FSH (p < 0.001) in 0, 1, 2, 4, 3, and 2 calves at 4, 8, 12, 24, 36, and 48 wk of age, respectively. The peak response of LH and FSH release to NMA was at 15 min posttreatment, and these peak responses were highest at 36 wk of age (p < 0.05). We suggest that neuroexcitatory amino acids, through NMDA receptors, are involved in prepubertal development of LH and FSH secretion in heifer calves.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the early postnatal period of heifer calves, circulating concentrations of LH are low; then between 10 and 22 wk of age there is a transient rise in secretion of LH [1, 2]. This early rise in circulating LH concentrations consists mainly of high-amplitude LH pulses and seems to have some stimulatory effects on ovarian follicular growth [2, 3]. After 22 wk of age, LH secretion is limited [2], but a progressive increase in frequency of LH pulses precedes sexual maturity [1, 2, 4–7]. Circulating concentrations of FSH show a similar pattern, except that the early rise appears to last longer, and FSH concentrations increase, then decrease in the few weeks prior to puberty [1, 2].

It has been suggested that an early increase in hypothalamic GnRH secretion is the main drive responsible for the early rise in gonadotropin release [7]. Endogenous opioids were shown to inhibit the secretion of gonadotropins during the immediate postnatal period in the heifer calf; however, their role is insignificant after the early rise in gonadotropin secretion [2]. Final maturation of gonadotropin secretion at puberty may be due to removal of some inhibitory influences at the hypothalamus or higher brain centers or the maturation of these areas [4, 8, 9].

The excitatory amino acids (EAAs) glutamate and aspartate are the major excitatory neurotransmitters in the central nervous system and play an important role in the regulation of gonadotropin secretion in many species [10]. The administration of an EAA analogue, N-methyl-D,L-aspartic acid (NMA), has been shown to stimulate LH secretion in rats [11, 12], sheep [13–15], monkeys [16, 17], and bull calves [18, 19]. Effects of NMA or other glutamate/aspartate agonists on pituitary release of LH and FSH are mediated by both N-methyl-D-aspartate (NMDA) and non-NMDA receptor subtypes, located throughout the central nervous system [20], and would involve the hypothalamic release of GnRH [21, 22]. Precocious puberty has been reported in response to NMA administration in rats [23] and monkeys [17]; however, there are no reports of such studies in heifer calves.

We hypothesized that EAAs could drive both the early postnatal and the prepubertal increase in gonadotropin secretion in heifer calves and that these periods of increased LH and FSH secretion could reflect increased hypothalamic sensitivity to EAAs. This was tested by measuring the response of LH and FSH secretion to the administration of NMA at critical points during development in heifer calves.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Ten spring-born, age-matched (± 5 days) Hereford heifer calves were maintained in a beef management system [2]. Calves were nursed at pasture until weaning at 21 wk of age, from which time they were kept in a paddock and were provided ad libitum with brome-alfalfa hay, water, and a ground concentrate ration of 21% ground barley, 51% ground hay, 22% ground straw, and 0.005% of a 1:1 calcium phosphorus mineral mix (w:w). Animals were weighed every 2 wk from birth until puberty. Animals were treated and handled according to the guidelines of the Canada Council of Animal Care.

Experimental Design

The calves were randomly assigned into two groups of five animals each. Five heifers received a bolus i.v. injection of NMA (4.7 mg/kg BW; Sigma Chemical Co., St. Louis, MO) dissolved in 0.9% NaCl at 4, 8, 12, 24, 36, and 48 wk of age. Body weights (mean ± SEM) at these ages were 59 ± 5, 82 ± 7, 108 ± 10, 181 ± 13, 248 ± 15, and 310 ± 18 kg, respectively. After 48 wk of age, animals started to reach puberty, and although we treated the remaining nonpubertal heifers at 52 and 56 wk of age, the number of animals was insufficient for statistical comparisons (control n = 2 and 2, treatment n = 3 and 2, at 52 and 56 wk, respectively); therefore, data up to 48 wk of age are presented. The dose of NMA was based on results from previous experiments in bull calves of the same breed, in which this dose of NMA induced an LH pulse as early as 4 wk of age (unpublished results), and in sheep [14]. Control heifer calves (n = 5) received vehicle (saline) at similar ages.

Blood samples (4–5 ml) were collected every 15 min for 10 h (0800–1800 h) at 4, 8, 12, 24, 36, and 48 wk of age, through jugular catheters, to characterize the pulsatile pattern of circulating concentrations of LH and FSH. NMA treatments were administered 1 h after the start of each period of frequent blood sampling (after the fifth sample). Jugular catheters (single-lumen polyvinyl chloride tubes; internal and external diameters 1.0 and 1.5 mm, respectively; Critchley Electrical Products Pty. Ltd., Silverwater BC, NSW, Australia) were inserted 1 day before each period of frequent blood sampling. During sampling, calves were housed loose in pens in a barn, with hay and water provided. Before weaning, cows were haltered with their calves. Blood samples were also taken every 2 wk, by jugular venipuncture, from 2 wk of age until puberty. Blood samples were allowed to clot for 12–18 h at room temperature, the clots were removed, and serum was centrifuged at 1500 x g for 15 min. The serum was poured off and stored at -20°C until analyzed.

Starting at 48 wk of age, to determine age at puberty, jugular blood samples were collected 2–3 times a week into sodium-heparinized vacutainers (10 ml; Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ) and centrifuged within 30 min of collection. Plasma was aspirated and stored at -20°C until analysis. Puberty was defined as the age at which serum concentrations of progesterone first exceeded 1.0 ng/ml, as an indication of first ovulation.

RIAs

Serum concentrations of LH and FSH were measured by double-antibody RIAs [2, 24]. LH concentrations are expressed in terms of NIDDK-bLH4 (Rockville, MD), and the sensitivity of the assay was 0.1 ng/ml (defined as the lowest concentration of unlabeled LH capable of displacing iodinated LH from the first antibody, p < 0.05). The range of the standard curve was from 0.06 to 8 ng/ml, and the intra- and interassay coefficients of variation were 8% and 13% (mean 0.25 ng/ml) and 5% and 11% (mean 0.72 ng/ml), respectively, for reference sera analyzed in replicate in every assay. FSH concentrations are expressed in terms of USDA-bFSH-I1. The sensitivity of the assay was 0.13 ng/ml. The range of the standard curve was 0.13 to 16 ng/ml. The intra- and interassay coefficients of variation were 6% and 8%, or 4% and 11%, respectively, for reference sera with mean FSH concentrations of 1.15 or 2.38 ng/ml, respectively.

Statistical Analysis

To characterize the pulsatile nature of serum concentrations of LH and FSH (including pulses in response to NMA) in samples collected every 15 min, the PC-Pulsar program (J. Gitzen and V. Ramirez, University of Illinois, IL) was used. From this analysis, mean and basal serum concentrations of LH and FSH and their pulse frequency and amplitude were determined. Pulses were identified using standard deviation criteria of height (G values) and duration [25]. The G values for LH pulse detection were G(1) 4.80, G(2) 3.00, G(3) 2.00, G(4) 1.80, and G(5) 1.50; for FSH pulse detection they were G(1) 13.2, G(2) 7.80, G(3) 5.76, G(4) 4.38, and G(5) 3.39. Basal concentrations were determined by subtraction of all hormone values included in the pulses from the periods of gonadotropin profiles. Values for LH and FSH less than assay sensitivity were regarded as being 0.1 ng/ml (assay sensitivity).

As the response of LH and FSH to NMA lasted no longer than 2 h and as NMA was given after 1 h of sampling, for statistical analysis the 10-h periods of frequent blood sampling were divided into two periods of the first 3 h and the remaining 7 h. Mean serum concentrations of LH and FSH over the first hour (5 pretreatment samples) were also analyzed for the effects of treatment group, age, and the interactions of treatment group by age using repeated measures ANOVA (RM-ANOVA; Sigma Stat for Windows Version 1.0; Jandel Corporation, San Rafael, CA) to ensure that heifers assigned to different treatment groups did not differ prior to NMA treatment. Mean gonadotropin concentrations, in the initial 3-h period, were then analyzed for the effects of treatment, time, and the interactions of treatment by time at each age using RM-ANOVA. Chi-square comparison was also used to detect any increase in gonadotropin secretion due to the NMA treatment. In order to do this, gonadotropin concentrations in the immediate posttreatment samples (sample 6) were given a score of 1 if they were higher than (at least 2 times assay sensitivity), or 0 if they were equal to or lower than, the immediate pretreatment sample (Chi-square Test for Trend, True Epistat; Epistat Services, Richardson, TX). The peak response of LH and FSH secretion to NMA treatment, as well as the percentage increase in LH and FSH secretion from pretreatment concentrations (sample 5) to the maximum concentrations following the NMA treatment, was analyzed for the effects of age using RM-ANOVA.

For the last 7 h of each period of frequent bleeding, RM-ANOVA was performed on the mean and basal concentrations of gonadotropins and their pulse frequency and amplitude in order to examine the effects of treatment, age, and the interactions of treatment by age. Age trends, however, are presented only for the control heifers. For control heifers only, mean and basal serum concentrations of gonadotropins and pulse frequency and amplitude, attained at each complete 10-h period of blood sampling, were also examined for the effects of age by RM-ANOVA in order to confirm the normal pattern of gonadotropin secretion during the prepubertal period in heifer calves.

LH and FSH concentrations in samples taken every other week were analyzed for the effects of treatment and age as well as their interactions using an RM-ANOVA. Comparison of age and weight at puberty as well as weight at birth was performed by one-way ANOVA (Sigma Stat).

In all analyses, if the main effects were significant, multiple comparisons were performed using the method of Student-Newman-Keuls for post-ANOVA comparisons, and only preplanned comparisons were made. All data are presented as mean ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
LH

Prior to administration of NMA, mean LH concentrations did not differ between the two groups at any age (p > 0.05, Table 1); however, in the control calves, mean LH concentrations increased from 24 to 48 wk of age (p < 0.05, Table 1). The number of animals responding to treatment increased with age (p < 0.01). Based on chi-square analysis, there was a response of LH concentrations to NMA, in comparison to the pretreatment values (p < 0.001, Fig. 1), in all treated calves older than 4 wk of age. At 4, 8, 12, 24, 36, and 48 wk of age, based on the PC-Pulsar analysis, 0, 3, 3, 4, 5, and 5 calves responded to NMA with a detectable LH pulse, respectively. Based on ANOVA, NMA treatment produced a significant increase in LH concentrations at 8, 12, 24, 36, and 48 wk of age as compared to pretreatment values (p < 0.05, Fig. 1). Peak response was seen at 15 min posttreatment (Fig. 1) and remained above pretreatment values for 15 min at 8, 12, and 24 wk of age but for 30 min at 36 and 48 wk of age (p < 0.05, Fig. 1). Compared to values in the control calves, LH concentrations were also higher at 15 min after NMA treatment at 8, 12, 24, and 48 wk of age (p < 0.05, Fig. 1). The peak response in LH release after NMA treatment increased from 4 to 36 wk of age (p < 0.05, Table 1). The percentage increase in LH secretion in response to NMA appeared to peak at 36 wk of age; however, this trend only approached significance level (p = 0.067, Table 1).


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  		TABLE 1. Mean (± SEM) serum concentrations of LH and FSH in samples taken every 15 min for 1 h prior to administration of NMA/saline, as well as peak response of LH and FSH and their percentage increase in response to NMA injection at various ages in heifer calves.



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  		FIG. 1. Mean (± SEM) serum concentrations of LH in samples taken every 15 min for the first 3 h of a 10-h sampling period from heifer calves at 4, 8, 12, 24, 36, and 48 wk of age. Heifers received NMA or saline after collection of the fifth blood sample (arrow). Values with an asterisk are different from control values at that time and age (p < 0.05). A cross represents values that differ (p < 0.05) from pretreatment values (in the NMA group).

During the last 7 h of the periods of frequent blood sampling, mean and basal LH concentrations, as well as the LH pulse frequency and amplitude, were not affected by the NMA treatment (p > 0.05). In the control calves, mean and basal LH concentrations appeared to be greatest at 48 wk of age; however, although an overall significant effect of age was seen (p < 0.04), no individual age group differences were seen (p > 0.05). LH pulse frequency in control calves increased significantly to 48 wk of age (p < 0.05). These trends were more pronounced when the entire 10-h period of frequent blood sampling of the control heifers was analyzed. Basal serum LH concentrations and LH pulse frequency were highest at 48 wk of age, and mean LH concentrations were greater at 48 wk of age compared to 8 and 24 wk of age (p < 0.05, Table 2).


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  		TABLE 2. Mean and basal serum concentrations of LH and FSH as well as LH and FSH pulse frequency and amplitude in samples collected every 15 min for 10 h in the control group of heifer calves at various ages.

When blood samples collected every other week were considered, there was no effect of treatment group (p > 0.05); therefore, the LH annual profiles for the two groups were pooled. Serum LH concentrations appeared to increase over the last 10 wk before puberty; however, although the overall effect of age was significant (p < 0.05, Fig. 2), there were no significant differences between individual age groups (p > 0.05). Retrospective comparison of LH values from 14 wk before to first ovulation gave a significant overall effect of age (p < 0.02); but again, although values at 2–4 wk before puberty appeared to be higher than earlier values, no significant differences were seen between individual age groups (p > 0.05).



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  		FIG. 2. Mean (± SEM) serum concentrations (n = 10) of a) LH and b) FSH in samples taken every 2 wk from 2 wk of age until puberty in heifer calves. Data for control and treatment calves did not differ (p > 0.05) and therefore were pooled. Superscript numbers are the number of animals included, as at later ages heifers that had reached puberty were deleted. Values with different superscript letters are significantly different (p < 0.05). Letter P shows the mean age at puberty (wk, mean ± SEM).

FSH

Mean FSH concentrations did not differ between the two groups, at any age (p > 0.05, Table 1), before NMA was administered. Based on chi-square analysis, there was a response of FSH concentrations to NMA in all treated heifer calves 8 wk of age and older in comparison to the pretreatment value (p < 0.001, Fig. 3). Based on the PC-Pulsar analysis, NMA injection induced a detectable FSH pulse in 0, 1, 2, 4, 3, and 2 calves of 5 treated calves at 4, 8, 12, 24, 36, and 48 wk of age, respectively. Serum FSH concentrations were increased for 15, 60, and 15 min after administration of NMA at 12, 36, and 48 wk of age, respectively, compared to pretreatment values (p < 0.05, Fig. 3). The peak response in mean serum concentrations of FSH after NMA treatment was highest at 36 wk of age (p < 0.05, Table 1). The percentage increase in FSH secretion in response to NMA increased from 4 to 24 wk of age (p < 0.05, Table 1).



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  		FIG. 3. Mean (± SEM) serum concentrations of FSH in samples taken every 15 min for the first 3 h of a 10-h sampling period from heifers at 4, 8, 12, 24, 36, and 48 wk of age. Heifers received NMA or saline after collection of the fifth blood sample (arrow). A cross represents values that differ (p < 0.05) from pretreatment values in the NMA group of heifers.

During the last 7 h of the periods of frequent blood sampling, mean and basal concentrations of FSH and FSH pulse frequency did not differ between the NMA-treated and control heifers (p > 0.05). NMA treatment resulted in a decrease in FSH pulse amplitude at 8 wk of age (p < 0.05, 0.37 ± 0.15 vs. 1.62 ± 0.25; treatment vs. control group, respectively). However, only 2 control and 3 treated calves had pulses at 8 wk of age. In control calves, FSH pulse amplitude was significantly higher at 8 wk of age compared to all other ages except 4 wk of age (p < 0.05, 0.76 ± 0.11, 1.62 ± 0.25, 0.55 ± 0.12, 0.52 ± 0.14, 0.52 ± 0.14, and 0.34 ± 0.14; at 4, 8, 12, 24, 36, and 48 wk of age, respectively). FSH profiles for the control calves during the entire 10-h periods of frequent blood sampling, however, did not reveal significant changes with age (p > 0.05, Table 2).

FSH concentrations in samples taken every other week did not differ between the NMA and control groups (p > 0.05); therefore, the annual FSH profiles of the two groups were combined. Serum FSH concentrations were highest at 8 wk of age and lowest at 48 wk of age (p < 0.05, Fig. 2). Retrospective comparison of the FSH concentrations over the period from 14 to 0 wk before the time of puberty did not show a significant trend (p > 0.05).

Age and Weight at Puberty

Age at puberty, as determined by measuring progesterone in blood samples taken 2–3 times a week, was not affected by treatment (p > 0.05, 55.2 ± 2.1 vs. 54.8 ± 1.9 wk; treatment vs. control group, respectively). Body weight at birth and puberty also did not differ between groups (p > 0.05; 35.8 ± 1 vs. 38.5 ± 3 kg and 349.6 ± 18 vs. 337 ± 30 kg; treatment vs. control, respectively).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
EAA neurotransmitters have been shown to cause LH release in several species [11, 13, 16] including bull calves ([18, 19] and our own unpublished results); however, the present report is the first in heifer calves.

The young postnatal heifer calf is responsive to exogenous GnRH [26]. However, in our study, heifer calves younger than 8 wk of age did not release LH or FSH in response to NMA treatment, and as animals grew, the responsiveness increased. These observations suggest that stimulation of gonadotropin secretion by NMA develops gradually in the young heifer calf but develops some time before first ovulation. It has been shown in immature rats that the LH response to NMA is also age related [12, 27], with a developmental pattern similar to that in the heifer. In the prepubertal female rat, release of LH by NMA develops between 10 and 15 days after birth and reaches its maximum responsiveness at about Day 27; first ovulation occurs after 4 wk of age [27]. It is possible that the LH-regulating systems responsible for responding to NMA stimulation are kept in check by inhibitory mechanisms in very young calves. This idea is supported by the finding that endogenous opioids actively inhibit LH secretion in 4-wk-old, but not older, heifer calves [2]. An interaction of endogenous opioids with EAA neurotransmitters was shown in young adult female mice; in that study, opioid antagonists did not stimulate LH release when injected alone, but they potentiated the LH response to NMA [28]. Therefore, the development of NMA sensitivity in heifers could be due to removal of an endogenous opioid brake.

In the present study, some animals had a robust (detectable by pulsar analysis) LH response to NMA injection as early as 8 wk of age, while some did not have such a marked response until they were 24 or 36 wk old. It is interesting to note that once each animal started to have a robust response to treatment, the animal consistently responded to the subsequent treatments at later ages. This indicates that the development of NMA induction of LH secretion is more dependent on physiological age than on the chronological age of the heifer.

It appeared that the stimulatory effects of NMA on gonadotropin release in prepubertal heifer calves were immediate and short, restricted to a single pulse within the 2-h period after treatment. The only longer-term effect of NMA treatment was the decreased FSH pulse amplitude at 8 wk of age. The reason for this is not clear, but it is possible that the FSH release induced by the treatment had decreased pituitary FSH reserves at this age; however, such an effect was not seen at other ages.

Although the response to NMA treatment involved both LH and FSH secretion, the responsiveness of LH and FSH to NMA was not identical. Differential responses of these gonadotropins to the stimulatory effects of NMA have been reported in rats and have been attributed to the differential involvement of NMDA/non-NMDA receptors in the release of LH and FSH [10, 29]. However, it is believed that the stimulatory effects of EAAs are exerted through the release of hypothalamic GnRH [22, 23]. In prepubertal heifers, pituitary response to GnRH in terms of LH and FSH release differs [30], and specific factors such as inhibin differentially regulate LH and FSH secretion [31, 32]. The more consistent short-term effect of NMA on LH as opposed to FSH release agrees with the differential effects of GnRH on LH and FSH release [30]. GnRH tends to more closely regulate pulsatile secretion of LH, whereas FSH secretion tends to respond more in the long term, not pulse by pulse [30]. However, it is also possible that there are EAA receptors in the pituitary cells [33, 34]. This kind of effect could differentially regulate LH and FSH secretion.

As a result of repetitive NMA injections, precocious puberty has been induced in rats [23] and monkeys [17] by stimulating GnRH neuronal activity. Although in the present study, NMA was a strong stimulator of LH release, neither age nor weight at puberty differed between the NMA and control heifers. The single injections with lengthy intervals between the treatments as used in our study would not have been expected to affect age and weight at puberty.

It has been suggested that gonadotropin concentrations are transiently increased prior to 22 wk of age in heifer calves [1, 2], but not all investigators have seen this early rise [8]. In the present study based on samples taken every 2 wk, serum concentrations of FSH declined from the early postnatal period, and little indication was seen of an early increase in LH secretion. In studies in which an early rise in LH secretion has been seen, it was suggested that this was due to an increase in LH pulse amplitude, but not frequency [2, 3]. Low-frequency high-amplitude pulses would be difficult to detect with blood samples taken every other week. When heifers in the present study were intensively bled, there was an indication of high-amplitude pulses at 8 wk of age in 2 heifers. Obviously, LH pulse frequency was very low at this time, and a longer period of bleeding may have allowed detection of high-amplitude pulses in more heifers. It is interesting that NMA sensitivity develops around and beyond the period when, as shown in previous studies, this initial increase in LH secretion occurs [1–3]. This is similar to the relationship between the early pattern of development of NMA responsiveness and LH secretion seen in female rats [27].

Increased LH pulse frequency resulting in higher mean LH concentrations was seen prior to first ovulation in this study, consistent with previous reports [2, 4]; LH concentrations seemed to reach their highest values 2–4 wk before puberty. The developmental FSH profile was variable, in agreement with earlier reports [2, 3], showing its highest values at 8 wk of age and declining prior to puberty. It was interesting to note that as in rats [35, 36], NMA responsiveness in terms of LH secretion was maximal some time before the final increase in LH secretion that occurs just prior to first ovulation. Therefore, sensitivity to EAAs would not appear to be involved with the onset of first ovulation in heifers and female rats.

Individual differences in response to NMA were noticeable through the present experiment, as was reported in sheep [37]. One heifer, for example, a few weeks before its first ovulation showed an LH surge in response to NMA injection (LH values rose up to 15 ng/ml and remained high for more than 5 h). EAAs have been shown to stimulate both types of LH secretion—pulsatile secretion and the preovulatory LH surge [10]. In experiments using peripubertal female rats it was concluded that NMA stimulation at a time close to sexual maturation releases endogenous EAAs from the hypothalamus, and this was directly correlated to the onset of puberty in these animals [36]. The authors also related this response to the maturation of the positive feedback effects of ovarian hormones on gonadotropin secretion [36]. Although the mechanisms by which EAAs exert their gonadotropic response are poorly understood, it was proposed that nitric oxide might be involved in the process and could convey the NMDA receptor signal to the GnRH neurons [38]. Central administration of a nitric oxide synthase inhibitor blocked the ability of NMA to induce LH release in rats [39]. We have shown that nitric oxide may also mediate NMA effects in heifer calves, as systemic blockade of nitric oxide production, prior to administration of NMA, significantly attenuated the LH response to NMA injection (unpublished results).

On the basis of the evidence presented in this study, we concluded that EAAs, through activation of NMDA receptors, appear to be involved in the regulation of LH and FSH release in heifer calves; their effects are age related, developing in the early postnatal period and reaching their maximum in the mid to late prepubertal period, sometime prior to first ovulation. In the present study, there was no clear evidence of an early postnatal increase in gonadotropin secretion, and clearly responsiveness of LH secretion to NMA does not cause the final increase in LH pulse frequency seen prior to first ovulation.


   ACKNOWLEDGMENTS
 
The authors wish to thank Susan Cook for her excellent technical assistance, Bill Kerr and his staff for care and management of the animals, and NIDDK and USDA for the provision of purified hormones. Ali Honaramooz would like to dedicate this paper to the memory of his father.


   FOOTNOTES
 
1 This study was supported by a Natural Sciences and Engineering Research Council Grant to N.C.R. Portions were presented at the 29th annual meeting of the Society for the Study of Reproduction, London, Ontario, Canada, July 27–30, 1996. A.H. was supported by the Iranian Ministry of Culture and Higher Education. Back

2 Correspondence: Norman C. Rawlings, Department of Veterinary Physiological Sciences, Western College of Veterinary Medicine, 52 Campus Drive, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 5B4, Canada. FAX: 306 966 7376; rawlings{at}sask.usask.ca Back

3 Current address: Department of Dairy and Poultry Sciences, P.O. Box 110920, University of Florida, Gainesville, FL 32611–0920. Back

Accepted: June 29, 1998.

Received: March 18, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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