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Serum Concentrations of Luteinizing Hormone, Growth Hormone, and Cortisol in Gilts Treated with N-Methyl-D,L-Aspartate During the Estrous Cycle or After Ovariectomy1 M. J. Estienne*,2, W. F. Hurlock*,3, and C. R. Barb† *Department of Agriculture, University of Maryland Eastern Shore, Princess Anne 21853 and †Animal Physiology Unit, ARS, USDA, Athens, GA 30613 ABSTRACT: The objective of this experiment was to determine the effects of n-methyl-d,l-aspartate (NMA), an agonist of the neurotransmitter glutamate, on circulating concentrations of LH, GH, and cortisol in gilts treated during the luteal ( n = 4 ) or follicular ( n = 4 ) phase of the estrous cycle, or after ovariectomy ( n = 4). Blood was sampled every 15 min for 10 h on each of two consecutive days. On the 1st d, two gilts from each group received i.v. injections of NMA (10 mg/kg BW) at h 4 and 6, and the remaining gilts received .9% saline (vehicle). The following day, gilts that had received NMA on the 1st d received vehicle, and gilts that had received vehicle on d 1 received NMA. All gilts received an i.v. challenge of GnRH (.1 mg/kg BW) at h 8 on each day. The NMA treatment increased ( P < .01) LH pulse frequency in luteal-phase gilts by 125%. In contrast, NMA decreased ( P < .05) mean concentrations of LH by 48% and suppressed ( P < .01) LH pulse frequency by 33% in ovariectomized gilts. No characteristics of LH secretion were affected ( P > .05) by NMA in follicular phase gilts. Serum LH concentrations for the 2-h period following GnRH were lower ( P < .05) in follicular-phase gilts than in ovariectomized gilts and were 1.15 ± .09 (mean ± SE), .81 ± .05, and .51 ± .17 ng/mL for ovariectomized, luteal-phase, and follicularphase gilts, respectively. Treatment with NMA increased circulating concentrations of GH by 334% ( P < .01) and cortisol by 77% ( P < .03) in all gilts. We suggest that the effects of NMA on LH release in gilts depend on the circulating steroidal milieu. In contrast, NMA evokes secretion of GH and cortisol irrespective of the reproductive status of treated gilts. Key Words: LH, Somatotropin, Hydrocortisone, Neurotransmitters, Gilts 1998 American Society of Animal Science. All rights reserved. Introduction Evidence suggests that glutamate, acting like a classical neurotransmitter, participates in neuroendocrine function and influences secretion of LH from the anterior pituitary gland in mammals (Brann et al., 1This research was supported by the Maryland Pork Producers Assoc., the USDA 1890 Capacity Building Grant Program, and Evans Allen Funds allocated to the Univ. of Maryland Eastern Shore. The authors wish to express sincere gratitude to S. Bishop, V. Cotton, and W. Douet and to B. Barrett, M. Stott, L. Baker, J. Popwell, and N. Whitley for their expert assistance with animal care and with laboratory analyses, respectively. The authors thank D. J. Bolt, ARS, USDA, Beltsville, MD, and A. F. Parlow, Harbor-UCLA Medical Center, Torrance, CA, for providing pituitary hormones, and antisera, respectively. 2To whom correspondence should be addressed: phone: 410-651-6194; fax: 410-651-6207. 3Current address: Wayne Farms, P.O. Box 470, Union Springs, AL 36089. Received December 30, 1997. Accepted April 10, 1998. J. Anim. Sci. 1998. 76:2162–2168 1996). Sesti and Britt (1992) reported that i.v. injections of n-methyl-d,l-aspartate ( NMA) , a potent agonist of glutamate, increased LH secretion in lactating sows and ovariectomized, estradiol-treated gilts. A stimulatory effect of NMA on LH secretion was also demonstrated in our laboratory, using prepubertal gilts as the experimental animals (Estienne et al., 1995). Consistent with a central site of action for NMA, administration of GnRH antisera to ovariectomized gilts abolished the ability of the compound to increase LH secretion (Sesti and Britt, 1992). In contrast, Barb et al. (1992) and Chang et al. (1993) reported that NMA was ineffectual in altering LH secretion in ovariectomized, estradiol-treated gilts and that it decreased LH release in ovariectomized, progesterone-treated gilts. These steroid treatments were used to establish, in ovariectomized gilts, circulating concentrations of steroids that mimicked those of the follicular (high estradiol) and luteal (high progesterone) phases of the estrous cycle. However, the effect of NMA on LH release in gilts during an actual estrous cycle has not been reported. 2162 ENDOCRINE RESPONSES TO N-METHYL-D,L-ASPARTATE Thus, in an effort to assess the role of glutamate in neuroendocrine regulation of pituitary hormone secretion, circulating concentrations of LH, GH, and cortisol were determined in gilts treated with NMA during the luteal and follicular phases of the estrous cycle or after ovariectomy. Materials and Methods Animals. The experiment was conducted at the University of Maryland Eastern Shore ( UMES) Swine Research and Education Facility in Princess Anne, and the experimental protocol was approved by the UMES Institutional Animal Care and Use Committee. Prepubertal Poland China × Yorkshire gilts ( n = 18) were given an i.m. injection of 400 IU of PMSG and 200 IU of hCG (P.G. 600, Intervet, Millsboro, DE). Seventeen gilts (94%) displayed standing estrus within 7 d after P.G. 600 treatment, and 13 gilts (72%) displayed a second estrus approximately 21 d later. Eight gilts that displayed a second estrus were then used for the experiment. The injections of P.G. 600 were staggered such that during the experiment gilts were in either the luteal ( 8 to 11 d after onset of estrus; n = 4 ) or follicular (17 to 20 d after onset of estrus; n = 4 ) phase of the estrous cycle. An additional four gilts were ovariectomized via midventral laparotomy 2 wk before the experiment. On the day before the experiment, all gilts were fitted with indwelling jugular vein catheters (Kraeling et al., 1982), which were used for collecting sequential blood samples and for administering NMA. At the time of the experiment, selected gilts were approximately 200 d of age and weighed an average of 122 kg. Gilts were individually penned (3.94 m2 totally slatted floor space/pen) in a passively ventilated, curtain-sided finishing barn. Average temperature was 20°C, and animals were exposed to the natural photoperiod for mid-August. Gilts were allowed ad libitum access to a fortified, corn-soybean meal pelleted diet (16% crude protein; Southern States Cooperative, Baltimore, MD) and to water. Experimental Protocol. Blood was sampled every 15 min for 10 h on each of two consecutive days. On the 1st d, two gilts from each group (i.e., luteal phase, follicular phase, or ovariectomized) received i.v. injections of NMA (Sigma Chemical Co., St. Louis, MO; 10 mg/kg BW) at h 4 and 6, and the remaining animals received .9% saline (vehicle). The dose of NMA used was previously shown to increase concentrations of LH in serum of prepubertal gilts (Estienne et al., 1995). On the following day, gilts that received NMA on the 1st d received vehicle, and gilts that had received vehicle on d 1 received NMA. All gilts received an i.v. challenge of GnRH (acetate salt; Sigma; .1 mg/kg BW dissolved in .9% saline) at h 8 on each day. Blood Handling Procedures and Radioimmunoassays. Blood samples were allowed to clot overnight at 4°C. 2163 Serum was collected and stored at −20°C until assayed. All samples were analyzed to determine serum LH concentrations with a specific RIA (Kesner et al., 1987). Intra- and interassay CV were 13.6 and 7.5%, respectively. Assay sensitivity was .15 ng/mL. Samples collected from h 0 to 8 were analyzed for GH using a previously reported procedure (Barb et al., 1991). Intra-and interassay CV were 2.1 and 1.3%, respectively, and assay sensitivity was .4 ng/mL. Samples collected from h 3 to 8 were analyzed for serum cortisol concentrations as previously reported (Barb et al., 1992). Intra- and interassay CV were 3.0 and 1.5%, respectively, and assay sensitivity averaged 1.0 ng/mL. The first samples collected on each day were analyzed for serum progesterone and estradiol concentrations using commercially available kits (Diagnostic Systems Laboratories, Webster, TX), validated for porcine serum in our laboratory. For the progesterone assay, parallelism was demonstrated by showing that estimates of progesterone concentrations were not influenced by volume of serum assayed (.4 to 25 mL). Recovery of .2, 7, or 9.1 ng of added progesterone averaged 92.6%. Assay sensitivity was .3 ng/mL, corresponding to 90% maximum binding of the label. The intraassay CV, determined by replicating a single serum pool containing 8.4 ng progesterone/mL twice in a single assay, was 13.6%. Parallelism of the estradiol assay was demonstrated by showing that estimates of estradiol concentrations were not influenced by volume of serum assayed (25 to 200 mL). Recovery of 4.7 or 280.1 pg of added estradiol averaged 101.2%. Assay sensitivity averaged 3.4 pg/mL, which corresponded to 90% maximum binding of the label. The intra- and interassay CV determined by replicating a single serum pool containing 5.1 pg of estradiol/mL four times in two assays were 11.7 and 22.0%, respectively. Statistical Analyses. Mean concentrations of LH, GH, and cortisol and the frequency and amplitude of LH pulses were calculated for the period before the first NMA or saline injection and the 4-h period after the first NMA or saline injection (i.e., h 4 to 8 ) for each gilt. A LH pulse was defined as an increment greater than the intraassay CV that occurred within 30 min after the previous nadir (Estienne et al., 1989). Pulse amplitude was defined as the difference between the pulse peak and the preceding nadir. Data were then subjected to ANOVA. The statistical model included treatment (NMA or saline), reproductive status (luteal, follicular, or ovariectomized), period (before or after initiation of NMA or saline injections), treatment × period, and treatment × period × reproductive status as possible sources of variation. If significant three-way interactions were detected, additional one-way ANOVA were conducted to determine the effects of period within treatment and reproductive status. When treatment × period was significant, additional one-way ANOVA were con- 2164 ESTIENNE ET AL. ducted to determine the effects of period within treatment (Gill and Hafs, 1971). Concentrations of LH that followed the GnRH challenge at h 8 were compared using ANOVA. The model included treatment, reproductive status, and treatment × reproductive status as possible sources of variation. Means for the three reproductive statuses were compared using Tukey’s studentized range test. Results One gilt that was classified as being in the follicular phase based on day of the estrous cycle had serum progesterone (6.55 ng/mL) and estradiol concentrations (below assay sensitivity) consistent with a luteal-phase gilt. This gilt was not included in the statistical analyses. Serum progesterone concentrations in the remaining luteal-phase animals were 8.11 ± 1.13 ng/mL (mean ± SE) and were undetectable in the follicular-phase and the ovariectomized gilts. Serum concentrations of estradiol were 5.38 ± 2.33 pg/ mL for follicular-phase gilts and were undetectable for luteal-phase and ovariectomized gilts. There was an effect ( P < .03) of treatment × period × reproductive status on mean LH concentrations (Figure 1). Before the injections, mean LH concentrations were .69 ± .07, .27 ± .02, and .24 ± .05 ng/mL for ovariectomized, luteal-phase, and follicular-phase gilts, respectively. Injections of saline had no effect ( P > .05) on mean LH concentrations in any group. Injections of NMA decreased ( P < .05) mean concentrations of LH in ovariectomized gilts by 48%. Treatment with NMA had no effect ( P > .05) on mean concentrations of LH in luteal- or follicular-phase gilts. The frequency of LH pulses before and after NMA or saline in ovariectomized, luteal-phase, and follicular-phase gilts is depicted in Figure 2. There was an effect ( P < .01) of treatment × period × reproductive status. Before the initiation of NMA and saline injections, LH pulse frequencies were 5.25 ± .16, .88 ± .12, and 1.52 ± .28 pulses/4 h for ovariectomized, luteal-phase, and follicular-phase gilts, respectively. Injections of saline had no effect ( P > .05) on LH pulse frequency in any group. Injections of NMA decreased ( P < .01) LH pulse frequency by 33% in ovariectomized gilts and increased ( P < .01) the frequency of LH pulses in luteal-phase gilts by 125%. An LH secretory episode identified as a pulse immediately followed every injection of NMA in luteal-phase gilts. In contrast, NMA had no effect ( P > .05) on LH pulse frequency in follicular-phase gilts. There were no effects ( P > .05) of treatment, treatment × period, or treatment × period × reproductive status on the amplitude of LH pulses. Amplitude of LH pulses was .36 ± .01 ng/mL for ovariectomized gilts, .55 ± .28 ng/mL for luteal-phase gilts, and .18 ± .02 ng/mL for follicular-phase gilts (effect of reproduc- Figure 1. Mean LH concentrations in serum of ovariectomized (OVX; n = 4), luteal phase (n = 4), and follicular phase (n = 3) gilts before (⁄; h 0 to 4) and during (◊; h 4 to 8) n-methyl-d,l-aspartate (NMA) or saline treatment. Blood samples were collected at 15-min intervals throughout the experiment, and NMA and saline were injected at h 4 and 6. Values are means ± SE. Injections of NMA decreased (P < .05; *) mean concentrations of LH by 48% in OVX gilts. In contrast, injections of NMA had no effect (P > .05) on mean LH concentrations in luteal or follicular phase gilts. tive status, P < .01). The LH pulse amplitude was higher before injections of NMA or saline (.43 ± .06 ng/mL) than after them (.32 ± .03 ng/mL; effect of period, P < .04). Serum LH profiles from h 0 to 8 for individual, NMA-treated ovariectomized, luteal-phase, and follicular-phase gilts are shown in Figures 3, 4, and 5, respectively. ENDOCRINE RESPONSES TO N-METHYL-D,L-ASPARTATE 2165 Figure 3. Serum LH concentrations in a representative, ovariectomized gilt. Blood samples were collected at 15-min intervals, and n-methyl-d,l-aspartate (NMA; 10 mg/kg BW) was injected i.v. at h 4 and 6 (represented by the vertical arrows). ovariectomized gilts and were 1.15 ± .09, .81 ± .05, and .51 ± .17 ng/mL for ovariectomized, luteal-phase, and follicular-phase gilts, respectively. Maximum serum concentrations of LH following GnRH were lower ( P < .05) in follicular-phase than in ovariectomized gilts and were 2.07 ± .16, 1.60 ± .09, and .77 ± .21 ng/mL for ovariectomized, luteal-phase, and follicular-phase gilts, respectively. Mean serum concentrations of GH and cortisol for gilts treated with NMA or saline appear in Table 1. There was no effect ( P > .05) of treatment × period × reproductive status for mean serum concentrations of Figure 2. Frequency of LH pulses in ovariectomized (OVX; n = 4), luteal phase (n = 4), and follicular phase (n = 3) gilts before (⁄; h 0 to 4) and during (◊; h 4 to 8) nmethyl-d,l-aspartate (NMA) or saline treatment. Blood samples were collected at 15-min intervals throughout the experiment, and NMA and saline were injected at h 4 and 6. Values are means ± SE. Injections of NMA increased (P < .01; **) LH pulse frequency in luteal phase gilts by 125% and decreased (P < .01; **) LH pulse frequency in OVX gilts by 33%. In contrast, NMA had no effect (P > .05) on LH pulse frequency in follicular phase gilts. There was an effect ( P < .01) of reproductive status but no effects ( P > .05) of treatment or treatment × reproductive status on serum concentrations of LH for the 2-h period following the GnRH challenge or on maximum LH levels after administration of GnRH. Serum LH concentrations for the 2-h period following GnRH were lower ( P < .05) in follicular-phase than in Figure 4. Serum LH concentrations in a representative, luteal phase gilt. Blood samples were collected at 15-min intervals, and n-methyl-d,l-aspartate (NMA; 10 mg/kg BW) was injected i.v. at h 4 and 6 (represented by the vertical arrows). 2166 ESTIENNE ET AL. Figure 5. Serum LH concentrations in a representative, follicular phase gilt. Blood samples were collected at 15-min intervals, and n-methyl-d,l-aspartate (NMA; 10 mg/kg BW) was injected i.v. at h 4 and 6 (represented by the vertical arrows). either hormone. There was, however, an effect of treatment × period for mean GH ( P < .01) and cortisol ( P < .04) concentrations. Injections of saline had no effect ( P > .05) on GH and cortisol levels. Treatment with NMA, however, increased serum concentrations of GH by 334% ( P < .01) and cortisol by 77% ( P < .03) in all gilts. Behavior of Gilts Following Administration of NMA. Gilts receiving injections of .9% saline displayed no noticeable behavioral changes. In contrast, injections of NMA caused vomiting. Discussion A group of amino acids that includes glutamate and aspartate and that are collectively referred to as the excitatory amino acids satisfy the main criteria for classification as neurotransmitters (van den Pol et al., 1996). Glutamate and aspartate, and receptors for these excitatory amino acids, are ubiquitously distributed in the brain and are in the hypothalamus and median eminence in many species (Petralia and Wenthold, 1996). There are several types of receptors that are stimulated by glutamate, and these are named according to their selective agonists. Receptor types include n-methyl-d-aspartate ( NMDA) , kainate, and d,l-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid ( AMPA) (Petralia and Wenthold, 1996). The excitatory amino acids may be the major neurotransmitters in the mammalian central nervous system and play a paramount role in the neuroendocrine control of anterior pituitary gland function. For swine, there is no information on the effects of kainate and AMPA receptor stimulation on secretion of LH. However, NMA is a potent agonist of the NMDA receptor and has been demonstrated to stimulate secretion of LH in lactating sows (Sesti and Britt, 1992, 1993, 1994); ovariectomized, estradiol-treated gilts (Sesti and Britt, 1992); and prepubertal gilts (Estienne et al., 1995). Consistent with these previous findings and in the current investigation, injections of NMA reliably evoked pulses of LH and, thus, increased the frequency of LH pulses in luteal-phase gilts. Increased secretion of LH following treatment of luteal-phase gilts with NMA is most likely a consequence of NMA-induced release of GnRH from the hypothalamus. Administration of GnRH antisera to ovariectomized gilts abolished the ability of the compound to increase LH secretion (Sesti and Britt, 1992), and NMA failed to alter basal or GnRHinduced gonadotropin release from rat or monkey pituitary glands in vitro (Tal et al., 1983). Nevertheless, NMA caused increases in LH secretion from pituitary cells collected from ovariectomized gilts and those with intact ovaries, which suggests that the compound may have subtle effects on the adenohypophysis (Barb et al., 1993). In contrast to the enhanced secretion of LH exhibited by NMA-treated luteal-phase gilts, injections of NMA had no effect on LH release in follicularphase gilts. There are at least two interpretations for this finding. First, follicular phase gilts may be less sensitive or completely insensitive to NMA with regard to the compound’s ability to stimulate GnRH and hence LH secretion. It is doubtful, however, that a higher dose of NMA could be used to evoke LH release, because the dosage used caused behavioral changes, including emesis. Alternatively, NMA might have stimulated GnRH secretion from the hypothalamus, but there was no corresponding increase in LH release because of effects of estradiol on the pituitary gland. In swine, gonadotrope responsiveness to GnRH is reduced by estradiol prior to the emergence of the LH surge (Cox Table 1. Mean serum concentrations (ng/mL) of GH and cortisol in gilts before and during treatment with n-methyl-d,l-aspartate (NMA) or with .9% salinea Item Before During GH NMAb Saline 1.10 ± .06 1.03 ± .06 4.77 ± .49 1.01 ± .06 Cortisol NMAc Saline 54.39 ± 9.84 38.17 ± 5.53 96.43 ± 10.95 43.72 ± 8.38 aValues bBefore cBefore are means ± SE. treatment vs during treatment differ ( P < .01). treatment vs during treatment differ ( P < .03). ENDOCRINE RESPONSES TO N-METHYL-D,L-ASPARTATE and Britt, 1982; Kesner et al., 1987, 1989). Following the GnRH challenge in this experiment, concentrations of LH were lower in early follicular-phase than in ovariectomized gilts, a finding consistent with the notion that pituitary responsiveness was compromised in follicular-phase animals. The lack of an effect of NMA on LH release in follicular-phase gilts is consistent with work by Barb et al. (1992), who demonstrated that LH secretion in ovariectomized, estradiol-treated gilts was unaffected by NMA. However, using a similar model and treatment regimen (dosages of estradiol and NMA, timing of intramuscular estradiol injection relative to i.v. NMA treatment, etc.), Sesti and Britt (1992) reported that NMA increased LH secretion. Perhaps undetermined genetic or environmental factors affect the ability of NMA to stimulate LH secretion. Consonant with previous reports (Barb et al., 1992; Chang et al., 1993; Popwell et al., 1996), in the current study, treatment with NMA decreased mean concentrations of LH and the frequency of LH pulses in ovariectomized gilts. Thus, in the absence of gonadal steroids, NMA suppresses gonadotropin secretion, which raises the possibility that in addition to their well-documented stimulatory effects, under appropriate conditions, excitatory amino acids may also suppress gonadotropin release. The reports by Barb et al. (1992) and Chang et al. (1993) that NMA also suppressed LH release in ovariectomized, progesterone-treated gilts seem incompatible with the current data, which revealed a stimulatory effect of NMA on LH release in lutealphase gilts. It should be noted, however, that in those previous studies (Barb et al., 1992; Chang et al., 1993) the steroid treatment regimen used was only marginally effective in suppressing gonadotropin secretion. As a consequence, it may not be surprising that when there was a high level of gonadotropin secretion before treatment, NMA injections ultimately resulted in a decrease in LH release. Previous investigations demonstrated that administration of NMA increased circulating concentrations of GH in ovariectomized gilts with or without steroid replacement therapy (Barb et al., 1992; Chang et al., 1993), in prepubertal gilts (Estienne et al., 1995), and in barrows (Estienne et al., 1996). Similarly, in the current experiment, GH secretion was evoked by NMA in ovariectomized, luteal-phase, and follicularphase gilts. The increase in GH secretion caused by NMA probably involves NMDA receptors and the hypothalamic secretion of GRF. The pretreatment of barrows with ketamine hydrochloride, an NMDA receptor antagonist, attenuated the ability of NMA to increase GH secretion (Estienne et al., 1996). Immunoneutralization of GRF in gilts (Barb et al., 1996) or barrows (Estienne et al., 1996) abolished NMAinduced GH secretion. However, Barb et al. (1993) 2167 reported that NMA stimulated GH release from pituitary cells collected from ovariectomized gilts and gilts in the luteal phase of the estrous cycle, but not from pituitary cells obtained from gilts in the follicular phase of the estrous cycle. Similar to previous studies conducted with ovariectomized gilts; ovariectomized, steroid-treated gilts; and barrows (Barb et al., 1992; Chang et al., 1993; Popwell et al., 1996), in the present study, NMA increased serum concentrations of cortisol in ovariectomized, luteal phase, and follicular phase gilts. The mechanism by which NMA increases cortisol secretion in swine has not been ascertained. However, it is likely that NMA stimulates secretion of corticotropinreleasing factor (CRF), which triggers pituitary release of ACTH and ultimately cortisol secretion. In rats, NMDA induced an increase in ACTH secretion (Farah et al., 1991). Moreover, Reyes et al. (1990) observed that intracerebroventricular infusion of CRF antiserum prevented NMA-induced cortisol secretion in ovariectomized rhesus monkeys. In summary, NMA had profound effects on circulating concentrations of LH, GH, and cortisol in swine. The role of excitatory amino acids in the physiological control of hormone secretion and in the neural mechanisms controlling growth, and reproductive events, such as the onset of puberty and the onset of estrus following weaning, warrants scrutiny. Implications Luteinizing hormone ( L H ) secretion is critical for reproductive events such as the onset of puberty and of estrus following weaning. Moreover, growth hormone ( G H ) stimulates growth and muscle accretion. Under appropriate conditions in the current study, nmethyl-d,l-aspartate stimulated LH, GH, and cortisol secretion in gilts. N-Methyl-d,l-aspartate mimics the actions of glutamate, an important neurotransmitter in the central nervous system. Thus, glutamate may have an important role in the control of hormone secretion in swine. This study provides basic information that will assist with the development of methods to accelerate growth and enhance reproduction in female swine. Literature Cited Barb, C. R., J. B. Barrett, G. B. Rampacek, and R. R. Kraeling. 1993. N-methyl-d,l-aspartate modulation of luteinizing hormone and growth hormone secretion from pig pituitary cells in culture. Life Sci. 53:1157−1164. Barb, C. R., R. M. Campbell, J. D. Armstrong, and N. M. Cox. 1996. Aspartate and glutamate modulation of growth hormone secretion in the pig: Possible site of action. Domest. Anim. Endocrinol. 13:81−90. Barb, C. R., G. M. Derochers, B. Johnson, R. V. Utley, W. J. Chang, G. B. Rampacek, and R. R. Kraeling. 1992. 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