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Effect of Gonadotropin-Releasing Hormone (GnRH) Pulse Frequency on Serum and Pituitary Concentrations of Luteinizing Hormone and Follicle-Stimulating Hormone, GnRH Receptors, and Messenger Ribonucleic Acid for Gonadotropin Subunits in Cows¹

Animal Science Department, Oklahoma Agricultural Experiment Station (J.A.V., R.P.W.), Stillwater, Oklahoma 74078-0425; the Department of Physiology and Pharmacology, Auburn University (T.D.B.), Auburn, Alabama 36849-5520; the Department of Physiology, Colorado State University (A.M.T., T.M.N.), Fort Collins, Colorado 80523

Address all correspondence and requests for reprints to: Dr. Robert P. Wettemann, Animal Science Department, Oklahoma State University, Stillwater, Oklahoma 74078. E-mail rpw{at}okway.okstate.edu

	Abstract

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Thirty-two nutritionally anestrous cows were used to determine the effect of the frequency of exogenous GnRH pulses on ovarian follicular growth, serum concentrations of LH and FSH, and concentrations of LH, FSH, GnRH receptors (GnRH-R), messenger RNA (mRNA) for GnRH-R, and mRNA for gonadotropin subunits in the pituitary. Cows were randomly assigned to one of four treatments: 2 µg GnRH infused (iv) continuously during 1 h, 2 µg GnRH infused during 5 min once every hour, 2 µg GnRH infused during 5 min once every fourth hour, or saline (control) for 13 days. Infusion of GnRH every hour increased LH concentrations in serum (P < 0.05), but FSH concentrations were not affected by GnRH infusion. Luteal activity (LA) was assessed by the presence of corpora lutea and/or serum progesterone greater than 1 ng/ml. Six of eight cows infused with GnRH every hour had LA by day 13, whereas only 25% of cows infused either continuously or with a pulse every fourth hour had LA by day 13. None of the control cows had LA during the experiment (P < 0.01). Concentrations of LH and FSH in the pituitary were significantly reduced when GnRH was infused hourly or continuously. Concentrations of common and FSHß mRNA were not influenced by treatment. However, continuous infusion of GnRH decreased (P < 0.05) LHß mRNA subunit. Concentrations of GnRH-R (P < 0.1) and GnRH-R mRNA (P < 0.05) were reduced when GnRH was infused continuously. We concluded that pulsatile secretion of LH is necessary for follicular growth and LA in beef cattle, and GnRH treatment differentially regulates LH and FSH gene transcription and serum concentrations of LH and FSH in cattle.

	Introduction

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GONADAL FUNCTION in beef cattle depends on pulsatile secretion of gonadotropins from the pituitary. The hypothalamus controls the secretion of gonadotropins by the pulsatile secretion of GnRH into the portal circulation of the pituitary (1, 2, 3). Continuous administration of GnRH resulted in desensitization of the pituitary gland in monkeys (4), sheep (5), and cows (6). Infusion of GnRH (2 µg) with a frequency of one pulse every hour induced luteal activity in nutritionally anestrous cows (7). Similarly, hourly infusion of calves with GnRH increased concentrations of LH in serum, and concentrations of messenger RNA (mRNA) for LHß and GnRH receptors (GnRH-R) in the pituitary (8). Secretion of LH and FSH in nutritionally anestrous cows is differentially regulated by the frequency and amplitude of GnRH pulses (9). The amplitude and duration of GnRH pulses can alter concentrations of , LHß, and FSHß mRNA in anterior pituitaries of rats (10).

Nutritionally anestrous cows have been used as a model to evaluate the secretion of serum LH, as the number and amplitude of LH pulses are reduced compared with those in cyclic cows (11, 12). Cows fed restricted diets released more LH in response to exogenous GnRH than cows fed moderate or high diets (13, 14) and had increased concentrations of GnRH in the stalk-median eminence (15). This suggests that decreased LH secretion in nutritionally restricted animals is due to reduced GnRH release from the hypothalamus.

The importance of pulse frequency of GnRH in the control of gonadotropin secretion and gene expression and of ovarian follicular growth in cows has not been determined. Therefore, the objectives of this experiment were to evaluate the effect of pulsatile vs. continuous infusion of nutritionally anestrous cows with GnRH on serum and pituitary concentrations of LH and FSH, ovarian activity, mRNA for gonadotropin subunits and GnRH-R, and concentrations of GnRH-R in the pituitary gland.

	Materials and Methods

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Animals and treatments
Thirty-two nonlactating, nonpregnant Hereford x Angus cows that exhibited normal estrous cycles were used. Cows that were 6 ± 2 yr of age and weighed 405 ± 15 kg were fed a restricted diet in a drylot until they became anestrus. Nutritional restrictions were initiated between the months of July and March, and cows were exsanguinated between November and August. The daily diet consisted of 2.7 kg prairie hay and 35 g of a mineral mix. When ambient temperatures were less than 0 C, each cow was fed an additional 1.4 kg of hay/day. These diets resulted in weight losses of approximately 1% of the initial body weight per week. Body weights and body condition score (BCS; 1 = emaciated and 9 = obese) (16) were determined every 14 days. Blood samples were taken weekly via venipuncture in 10-ml tubes containing EDTA to prevent clotting. Samples were placed on ice and centrifuged (3000 x g for 20 min) within 4 h, and plasma was decanted and stored at -20 ± 2 C until progesterone was quantified. Concentrations of progesterone less than 1 ng/ml for 3 consecutive weeks were used to identify anestrous cows. Cows became anovulatory after approximately 120 days of feed restriction and at a BCS of 4.

Within 3 weeks after the onset of anestrus, cows were confined in individual stalls in a barn at 21 ± 4 C and 50 ± 10% relative humidity with 14 h of light/day. To maintain the nutritional anestrous state, each cow was fed 5.5 kg of prairie hay and 35 g of a mineral mix at 0900 h daily. A polyvinyl jugular cannula (id, 1.68 mm; od, 2.39 mm; BB 317 v11, Bolab, Lake Havasu City, AZ) was inserted into each external jugular vein 2 days before treatment to facilitate simultaneous infusion and blood collection. Experimental procedures were approved by the Oklahoma State University animal care and use committee.

Cows were randomly assigned to one of four treatments (n = 8/treatment): GnRH (2 µg; Sigma Chemical Co., St. Louis, MO) infused (iv) continuously during 1 h (GnRH-C), GnRH (2 µg) once every hour (GnRH-1), GnRH (2 µg) once every 4 h (GnRH-4), or saline (control; 1.8 ml/h). Pulsatile infusions (GnRH-1, GnRH-4 and control) were conducted with a Harvard infusion pump (model 931, Harvard Infusion/Withdrawal Pump, South Natick, MA). The pump was controlled by an automatic timer (model CD-4, ChronTrol, Lindburg Ent, San Diego, CA). This pump-timer unit was calibrated to deliver the pulse of saline or GnRH over 5 min. Continuous infusion was conducted with a peristaltic pump (Manostat Pump, Fisher Scientific, Pittsburgh, PA) that delivered 34 ml/h. Heparin (1 USP/ml) and penicillin (50 U/ml) were added to sterile saline to prevent clotting and bacterial contamination of cannulas during infusion.

Treatments were initiated on day 0 at 0800 h and continued through 0800 h on day 13. Blood serum samples (10 ml) were collected at 10-min intervals for 8 h commencing the day before treatment (day -1) and just before the pulse of GnRH or saline at 0800 h on days 0, 2, 4, and 12. Samples were allowed to clot at 21 ± 4 C for 1 h and then stored at 4 C for 20 h. Samples were centrifuged (3000 x g for 20 min), and serum was decanted and stored at -20 ± 2 C until LH and FSH concentrations were determined. In addition, blood samples were taken daily from days -1 through 12 in 10-ml tubes containing EDTA. Samples were placed on ice and centrifuged (3000 x g for 20 min) within 1 h, and plasma was decanted and stored at -20 ± 2 C until progesterone and estradiol concentrations were quantified.

Ultrasonography
Ultrasound examinations were performed with an Aloka 500V ultrasound scanner equipped with a 7.5-MHz transducer (Corometrics Medical Systems, Wallingford, CT) designed for intrarectal examination. Both ovaries of each cow were evaluated on days -1, 1, 3, 5, 7, 9, 11, and 13. The number and size of follicles larger than 2 mm and the presence of corpora lutea (CL) were recorded. The number of follicles was categorized as small (2–6 mm), medium (6–8 mm), and large (> 8 mm). Luteal activity was assessed by the presence of a CL and/or serum progesterone concentrations greater than 1 ng/ml for 3 consecutive days after day 1 of treatment.

Tissue collection
Within 4 h of the cessation of treatments on day 13, cows were exsanguinated. Ovaries were evaluated to assess the numbers and sizes of CL and follicles. Pituitary glands were removed and placed on ice within 10 min. Glands were trimmed and hemisected midsagitally, and the posterior lobe was removed. The anterior lobe was weighed and frozen at -72 C within 25 min after exsanguination. Concentrations of LH (micrograms per mg tissue), FSH (micrograms per mg tissue), and GnRH-R (femtomoles per mg protein) were measured after pituitary samples were thawed and homogenized in buffer (10 mm Tris, 1 mm CaCl₂, and 0.25 M sucrose, pH 7.0). Tissue was initially homogenized with two 5-sec bursts in a Tissue Tearor (Biospec, Bartlesville, OK), followed by homogenization first in a ground glass homogenizer and then in a Dounce glass homogenizer (Kontes Co., Vineland, NJ) at 4 C. The homogenate was diluted and centrifuged at 16,000 x g for 15 min at 4 C. The supernatant was retained and frozen for analyses of LH and FSH concentrations. The homogenate was resuspended in buffer to assay GnRH-R.

Analyses of mRNA
For analyses of pituitary mRNAs, polyadenylated [poly(A)⁺] RNA was isolated from half of each pituitary gland by binding to oligo(deoxythymidine)cellulose (17). The integrity of this RNA was verified by hybridization to radiolabeled ovine GnRH-R complementary DNA (cDNA) in Northern blot analysis as previously described (18). Four GnRH-R mRNA transcripts were observed at 5.6, 3.2, 2.5, and 1.6 kilobases. These findings are in good agreement with those observed in bovine pituitary RNA using a homologous cDNA probe (19). Steady state concentrations of pituitary mRNAs were determined by slot blot analyses. Either 0.5 (for - and LH ß-subunits) or 1.0 µg (for FSH ß-subunit and GnRH-R) poly(A)⁺ RNA was applied in duplicate to nylon membranes (Hybond, Amersham, Arlington Heights, IL) and cross-linked with UV light (Stratagene, La Jolla, CA). Slot blot membranes were hybridized at 42 C for 24 h to cDNAs encoding bovine -subunit (20), bovine LH ß-subunit (21), bovine FSH ß-subunit (22), and ovine GnRH-R (18). Each cDNA was radiolabeled using the random hexamer priming method (Boehringer Mannheim, Indianapolis, IN). Hybridization buffer consisted of 50% formamide, 0.5% SDS, 0.1 mg/ml denatured salmon sperm DNA, 0.02 M piperazine-N,N'-bis[2-ethane-sulfonic acid] (PIPES), 0.8 M NaCl, and 0.002 M EDTA. Final washes of membranes were performed in 0.5 x SSC (standard sodium citrate)-0.1% SDS at 65 C for -subunit, 0.5 x SSC-0.1% SDS at 42 C for FSH ß-subunit, and 1 x SSC-0.1% SDS at 42 C for LH ß-subunit and GnRH-R. To adjust for differences in loading among RNA samples, blots were stripped of gonadotrope-specific cDNAs by washing in 50% formamide and 10 mm NaPO₄ at 65 C for 1 h and reprobed with radiolabeled rat -tubulin cDNA (23) under conditions identical to those described above, except that the final wash was performed at 0.1 x SSC-0.1% SDS at 65 C. Membranes were exposed to Hyperfilm-MP (Amersham) for 0.5–4 days. Autoradiographs were analyzed with a scanning densitometer (Hoefer Scientific Instruments, San Francisco, CA). Results are expressed as arbitrary densitometric units.

Concentrations of GnRH-R
Receptors for GnRH were quantified as described by Nett et al. (24) with modifications. Briefly, buserelin ([D-Ser(tBu)⁶,Pro⁹NHEt]GnRH, a gift from Hoescht Roussel Pharmaceuticals, Summerville, NJ; 5 µg) was radioiodinated in the presence of 2 mCi Na¹²⁵I and 0.5 µg chloramine-T in a sodium phosphate buffer (150 mM Na PO₄, pH 7.4) for 3 min at room temperature (25) and used as ligand. Free ¹²⁵I was separated from [¹²⁵I]buserelin and noniodinated buserelin by elution on a Sephadex G-25 column eluted with 50 mm acetic acid containing 0.1% BSA. The resulting [¹²⁵I]buserelin is typically 40–50% bindable to excess receptor with a specific activity, estimated by self-displacement, to be 1100 Ci/mM (26).

Assay buffer consisted of 10 mM Tris, 1 mm CaCl₂, and 0.3% (wt/vol) BSA, pH 7.0. A standard curve was generated using several quantities of GnRH-R (0.32–16.8 fmol) from a pool of bovine pituitary membrane incubated with a constant quantity of [¹²⁵I]buserelin (4.8 fmol). Sample tissue from each cow was incubated with the same concentration of ¹²⁵I as the standard curve. The number of GnRH-R used for the standard curve was determined by Scatchard analysis (27). Binding of [¹²⁵I]buserelin to sample tissues was directly compared to the standard curve to determine the number of GnRH-R in sample tissues. All assay volumes were 150 µl. Steady state binding of [¹²⁵I]buserelin was attained by 2 h at 4 C and was maintained for at least 12 h. All samples were incubated for 4 h at 4 C. To terminate the assay, 3 ml ice-cold assay buffer were added to each tube followed by centrifugation at 16,000 x g for 15 min at 4 C. After centrifugation, the supernatant was decanted, and the radioactivity associated with the membrane pellet was quantified by solid scintillation counting.

Protein concentrations were determined using Coomassie Plus Protein regent (Pierce Chemical Co., Rockford, IL). Results are expressed in femtomoles per mg protein.

Hormone assays
Concentrations of LH (7) in serum (250 µl) and pituitary tissue (250 µl of a 1:10,000 dilution) were measured in duplicate by RIA, and NIH LH-B9 was the standard. The inter- and intraassay coefficients of variation were 11% and 20% (n = 25).

FSH was quantified by a RIA similar to that described by Bolt and Rollins (28) and Crowe et al. (29), using antiovine FSH serum (NIDDK-oFSH-I-1), bovine FSH (USDA-bFSH-I-2) for standards and ovine FSH (USDA-oFSH-19-SIAFP-I-2) labeled with ¹²⁵I. Sodium phosphate buffer (NaPO₄; 25 µl; 0.5 M; pH 7.5), ¹²⁵I (300 µCi in 3 µl H₂O), and chloramine-T (7.5 µg in 3.5 µl NaPO₄; 0.05 M; pH 7.5) were added to a vial that contained 10 µg ovine FSH. After 60 sec of incubation, the reaction was terminated with 50 µg sodium metabisulfite in 12.5 µl NaPO₄ (0.05 M; pH 7.5). A Bio-Gel P-60 column (Bio-Rad Laboratories, Richmond, CA; 50–100 mesh) was used to separate [¹²⁵I]FSH from free ¹²⁵I. Antiserum against FSH was diluted 1:80,000 in PBS containing a 1:400 dilution of normal rabbit serum (Sigma Chemical Co., St. Louis, MO), and 200 µl were added to tubes (12 x 75 mm) containing standards (6.25 pg to 1.6 ng) in 500 µl PBS with 0.1% gelatin (PBS-G) or unknown serum (250 µl serum plus 250 µl PBS-G). After incubation for 24 h at 21 ± 3 C, 100 µl [¹²⁵I]FSH (15,000 cpm in PBS-G) were added to each tube, and tubes were incubated for 24 h at 21 ± 3 C. Then, 200 µl sheep antirabbit -globulin (1:30 dilution) were added. After incubation for 24 h at 4 ± 2 C, 2.5 ml PBS (4 C) were added to each tube. Tubes were centrifuged for 30 min (2,500 x g), the supernatant was aspirated, and radioactivity in the precipitate was quantified with a -counter. The addition of 5 ng FSH to 1 ml serum resulted in 90% recovery (n = 30). Cross-reactivity with PRL (NIH-P-B4) and LH (NIH-LH-B4) was less than 1%. Cross-reactivity with GH (NIH-GH-B17) and TSH (NIH-TSH-B7) was less than 2%. When different volumes of serum were assayed, concentrations were parallel to the standard curve. Inter- and intraassay coefficients of variation were 1.4% and 13.0%, respectively.

Progesterone was quantified using a solid phase RIA (Coat-A-Count progesterone kit, Diagnostic Products Corp., Los Angeles, CA). The addition of 5 ng progesterone to 1 ml plasma resulted in 92% recovery (n = 22). When different concentrations of plasma were assayed, concentrations were parallel to the standard curve. Inter- and intraassay coefficients of variation were 3.7% and 7.2%, respectively (n = 22).

Concentrations of estradiol were quantified by a RIA similar to that described by Evans et al. (30) using a Serono Estradiol MAIA assay kit (Biodata SpA, Montecelio, Italy) with the following modifications. Duplicate standards or unknown plasma samples were extracted in glass tubes (12 x 75 mm) with 2 ml ethyl acetate, and aliquots (1 ml) of the solvent layer were evaporated under nitrogen gas. The sensitivity was 0.6 pg estradiol/ml plasma. The addition of 7 pg estradiol to 1 ml plasma resulted in 95% recovery (n = 5). When different volumes of plasma or extract were assayed, concentrations were parallel to the standard curve. Inter- and intraassay coefficients of variation for a sample that contained 7 pg/ml estradiol were 16.1% and 16.9%, respectively (n = 5).

Statistical analyses
Split plot analyses of variance were used to determine the effect of treatment over days on serum LH and FSH concentrations and numbers of ovarian follicles and CL. Cow within treatment was the error term used to test treatment effects, and cow within treatment x day was used as the error term to test day of treatment and the interaction of day with treatment. To minimize the variation among cows, LH and FSH concentrations on day -1 were used as a covariate to analyze LH and FSH concentrations. Split plot analyses of variance were used to determine the effect of treatment and the presence of large follicles (>8 mm) on estradiol concentrations and the effect of treatment and the presence of a CL on progesterone concentrations. The variances of the densitometric units for mRNA were proportional to the treatment means; therefore, the natural logarithm of the unit was used (31). ANOVAs were used to determine the effect of treatment on concentrations of mRNA for common -, LH ß-, FSH ß-subunits and GnRH-R, and concentrations of GnRH-R in the pituitary. Luteal activity among treatments was compared by Fisher’s exact test (31).

	Results

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Gonadotropin secretion
Serum concentrations of LH and FSH on day -1 averaged 3.30 ± 0.05 and 0.46 ± 0.01 ng/ml, respectively, and were not different among treatments. There was a significant treatment effect on serum LH, but not on serum FSH, during the 12 days of treatment (Fig. 1). Day of treatment did not influence serum concentrations of LH or FSH. Cows infused with a pulse of GnRH every hour had greater concentrations of LH during the infusion period than control cows (P < 0.05). Concentrations of serum LH in cows infused every fourth hour with GnRH did not differ from those in control cows. The response to continuous GnRH was not different from that in control or GnRH-1 cows.

View larger version (16K): [in this window] [in a new window]   Figure 1. Serum concentrations of LH and FSH in anestrous cows (n = 8/treatment) that were treated for 13 days with 2 µg GnRH infused (iv) continuously during 1 h (GnRH-C), during 5 min once every hour (GnRH-1), or during 5 min once every fourth hour (GnRH-4) or with saline (control). Values are the mean ± SE LH and FSH levels on days 0, 2, 4, and 12. Different letters within a hormone indicate a significant differerence (P < 0.05).

View larger version (18K): [in this window] [in a new window]   Figure 2. Serum concentrations of estradiol in anestrous cows (n = 8/treatment) that were treated for 13 days with 2 µg GnRH infused (iv) continuously during 1 h (GnRH-C), during 5 min once every hour (GnRH-1), or during 5 min once every fourth hour (GnRH-4) or with saline (control). Values are the mean ± SE estradiol concentration during the experiment in cows with small and medium follicles (2–8 mm) and cows with large follicles (> 8 mm). Different letters indicate a significant difference (P < 0.0001).

There was a treatment x day x presence of CL effect (P < 0.003) on concentrations of progesterone in plasma. None of the control cows or cows without a CL had concentrations of progesterone greater than the sensitivity of the assay (0.1 ng/ml). Average concentrations of progesterone during days 5–12 of treatment for cows with a CL were greater (P < 0.01) when cows were treated with a pulse of GnRH every hour (1.2 ± 0.2 ng/ml; n = 6) or treated with GnRH continuously (1.3 ± 0.3 ng/ml; n = 2) than when cows were given a pulse of GnRH every fourth hour (0.3 ± 0.1 ng/ml; n = 2).

Anterior pituitary determinations
Anterior pituitary glands weighed 1.85 ± 0.05 g and did not differ among treatments (P > 0.18). However, concentrations of LH and FSH in the pituitary gland were affected by treatments (Fig. 3). Control cows had greater concentrations of LH in pituitaries (P < 0.05) compared with GnRH-C- and GnRH-1-treated cows. Concentrations of FSH were greater in control cows (P < 0.05) than in those receiving the other treatments.

View larger version (21K): [in this window] [in a new window]   Figure 3. Concentrations of LH and FSH in the pituitary gland of anestrous cows (n = 8/treatment) that were treated for 13 days with 2 µg GnRH infused (iv) continuously during 1 h (GnRH-C), during 5 min once every hour (GnRH-1), or during 5 min once every fourth hour (GnRH-4) or with saline (control). Values are the mean ± SE LH and FSH levels on day 13. Different letters within a hormone indicate a significant difference (P < 0.05).

View larger version (22K): [in this window] [in a new window]   Figure 4. Concentrations of GnRH-R and GnRH-R mRNA in the pituitary gland of anestrous cows (n = 8/treatment) that were treated for 13 days with 2 µg GnRH infused (iv) continuously during 1 h (GnRH-C), during 5 min once every hour (GnRH-1), or during 5 min once every fourth hour (GnRH-4) or with saline (control). Values are the mean ± SE concentrations of GnRH-R and GnRH-R mRNA on day 13. Different letters within GnRH-R (P < 0.1) and GnRH-R mRNA (P < 0.05) indicate a significant difference.

View larger version (22K): [in this window] [in a new window]   Figure 5. Steady state concentrations of common -, LH ß-, and FSH ß-subunit mRNAs in the pituitary glands of anestrous cows (n = 8/treatment) that were treated for 13 days with 2 µg GnRH infused (iv) continuously during 1 h (GnRH-C), during 5 min once every hour (GnRH-1), or during 5 min once every fourth hour (GnRH-4) or with saline (control). Values are the mean ± SE concentrations of common -, LH ß-, and FSH ß-subunit mRNAs on day 13. Different letters within each subunit indicate a significant difference (P < 0.05).

	Discussion

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The number of small follicles was greater in control cows than in GnRH-treated cows. However, the number of medium follicles was not different among treatments. Stimulation of the ovary by the release of gonadotropins in cows infused with GnRH might have induced growth of follicles and decreased the available pool of small follicles.

Plasma concentrations of estradiol in control cows during the 13 days averaged 0.3 ± 0.1 pg/ml. Cows infused with GnRH once every hour that had follicles greater than 8 mm (n = 6) had an average of 5.4 ± 0.8 pg/ml estradiol. Concentrations of estradiol ranged from 1.5–4.2 pg/ml (luteal and follicular phases, respectively) during the estrous cycle of cows (32, 33), indicating that GnRH-1 cows had estradiol concentrations similar to those during the late follicular phase in cyclic cows. However, GnRH-4, GnRH-C, and control cows with a large follicle (>8 mm) had an average of 0.8 ± 0.2 pg/ml estradiol in plasma, and cows with follicles from 3–8 mm had 0.7 ± 0.1 pg/ml, indicating that these large follicles synthesized a similar amount of estradiol compared with small to medium follicles. Treatment of cows with a pulse of GnRH once every hour stimulated estradiol synthesis in large follicles, and greater amounts of estradiol were secreted.

Two of eight cows that received a pulse of GnRH every fourth hour had a CL by day 13, whereas six of eight cows that received a pulse of GnRH every hour had a CL by day 13. Similarly, nutritionally anestrous cows treated with hourly infusions of 2 µg GnRH for 14 days had concentrations of progesterone greater than 1 ng/ml within 5 days of treatment, and by day 14, concentrations of progesterone were greater than 2 ng/ml (7). In the present experiment, LH concentrations were significantly greater in GnRH-1 cows than in cows given a pulse of GnRH every fourth hour, GnRH continuously, or saline. As LH is luteotropic in cattle (34, 35), the presence of luteal tissue in 75% of the cows was probably due to enhanced GnRH-stimulated LH secretion.

The total amount of GnRH that was given during treatment in GnRH-C cows was the same as that in GnRH-1 cows. However, 75% of the GnRH-1 had luteal activity, whereas only 25% of the GnRH-C cows developed luteal tissue. The presumption of a pulsatile release of GnRH in monkeys was first suggested by Dierschke et al. (36). Rodriguez and Wise (3) found that GnRH was released in a pulsatile manner when hypophyseal-portal blood was collected from calves. Results from the present experiment indicate that in the cow, hourly pulses of GnRH are needed to attain significant physiological effects at the ovary. However, we cannot rule out the possibility that the greater ovarian response in cows given a pulse of 2 µg GnRH in 5 min compared with cows given 2 µg in 60 min is not due to the concentration of GnRH that is achieved in cows when the same amount of GnRH is given in a shorter period.

Anterior pituitary weights were similar to those observed in thin beef cows (14), cyclic beef cows (37), and dairy cows (38). Results from this experiment indicate that infusion with GnRH did not influence pituitary weight. Likewise, nutrient intake and BCS, which influence LH secretion, did not affect pituitary weight in cows (14) and heifers (37).

Concentrations of LH (adjusted to the NIH-LH-S1 reference preparation) in the anterior pituitary of control cows were similar to those observed during proestrus (39), metestrus (14), and the luteal phase (40) in beef cows. After 13 days of hourly or continuous infusion of GnRH (2 µg/h), cows had 68% and 64% less LH in the pituitary compared with control cows. Reduced concentrations of LH in pituitaries of GnRH-1 cows may be related to LH release associated with ovulation and/or formation of luteal tissue. Cupp et al. (41) found that cows only had 30% as much LH after the ovulatory surge compared with concentrations during proestrus. Reduced concentrations of LH in GnRH-C cows compared with control cows could be related to desensitization of the pituitary due to continuous exposure to GnRH (6).

Concentrations of FSH in the anterior pituitary were significantly reduced by infusing GnRH. When 2 µg GnRH were given once every fourth hour, concentrations of LH in the pituitary were not significantly different from those in control cows; however, GnRH-4 cows had reduced concentrations of FSH compared with control cows. This suggests a differential effect of GnRH on the regulation of LH and FSH release and/or synthesis in the bovine. Wildt et al. (42) also found that reducing the frequency of exogenous GnRH pulses to monkeys, resulted in reduced concentrations of LH and greater FSH secretion in serum.

This is the first report on the effect of continuous administration of exogenous GnRH on concentrations of GnRH-R mRNA in the bovine pituitary gland. Our major findings are that pulsatile GnRH does not influence concentrations of GnRH-R or GnRH-R mRNA, but continuous infusion of GnRH dramatically reduces the concentrations of unoccupied GnRH-R and GnRH-R mRNA in the pituitary gland.

Homologous up- and down-regulation of pituitary GnRH-R has been demonstrated in several mammalian species (43, 44). Hourly infusion of GnRH in 1-week-old calves resulted in increased numbers of GnRH-R compared with those in saline-infused animals (8). Similarly, GnRH pulses given at 30-min intervals for 12–24 h up-regulated GnRH-R gene expression in ovariectomized rats that were treated with an -adrenergic antagonist (45). Steroids can also have an important role in the regulation of the number of GnRH-R. In sheep, estradiol can exert a direct effect on concentrations of GnRH-R (46) and on GnRH-R mRNA (47). However, during the preovulatory period, an increase in concentrations of GnRH-R occurred when concentrations of progesterone decreased and before concentrations of estradiol increased (18). In the present experiment, two of eight cows in the GnRH-1 treatment did not have a CL on day 13. Concentrations of progesterone and estradiol in plasma and GnRH-R in the pituitary on day 13 were less than 0.1 ng/ml, 12.2 ± 3.1 pg/ml, and 13.0 ± 4.3 fmol/mg protein, respectively, in the two cows without luteal tissue. Concentrations of progesterone and estradiol and the number of GnRH-R in the other six cows that had a CL on day 13 were 1.4 ± 0.8 ng/ml, 0.2 ± 0.1 pg/ml, and 4.9 ± 0.8 fmol/mg protein, respectively. Although the number of animals is limited, these data indicate that up-regulation of GnRH-R occurs in cows in the absence of progesterone and in the presence of estradiol in plasma.

In vitro treatment of sheep pituitary cells with a GnRH agonist, down-regulated the number of GnRH-R (48). Similarly, the addition of 1 µm GnRH to T₃-1 pituitary cells resulted in down-regulation of GnRH-R-binding sites (49). Down-regulation of concentrations of GnRH-R and GnRH-R mRNA was observed in the present experiment when GnRH was infused continuously. Two cows in the GnRH-C treatment group had luteal tissue on day 13, and the number of GnRH-R in these cows averaged 3.9 ± 1.5 fmol/mg protein. Concentrations were similar (2.8 ± 0.8 fmol/mg of protein) in the six cows that did not have luteal tissue. This suggests that during continuous infusion with GnRH, it is unlikely that up-regulation of GnRH-R in the absence of progesterone can occur.

Concentrations of common - and FSH ß-subunit mRNAs were not affected by treatment, whereas continuous infusion of GnRH caused a reduced concentration of LHß mRNA compared with the other treatments. Cows given GnRH every hour had concentrations of LHß mRNA that were not different from those in control or GnRH-4 cows. As six of eight of the cows receiving GnRH-1 treatment had luteal activity at exsanguination, the stage of the cycle may have influenced pituitary LHß mRNA. The time that gonadotropin subunit mRNA are quantified in pituitaries, relative to the onset of GnRH treatment, may also influence steady state concentrations. The amount of mRNA for LHß in the pituitary of ovariectomized ewes was influenced by the number of days exposed to estradiol (50). Ovariectomized, estradiol-treated rats (10) and orchidectomized, testosterone-implanted rats (51, 52) responded to pulsatile GnRH with increased , LHß, and FSHß mRNA. In prepubertal male cattle, hourly infusion of GnRH increased concentrations of common and LHß mRNA compared with those in control animals (8).

In conclusion, pulsatile secretion of LH is necessary for follicular growth in cows, and GnRH differentially regulates gene transcription and the secretion of LH and FSH. Pulsatile GnRH treatment that stimulated follicular growth and luteal activity did not significantly alter serum FSH secretion or FSHß mRNA concentrations. These data are compatible with the hypothesis that FSH is not limiting in nutritionally induced anestrous cows.

	Acknowledgments

 
The authors gratefully acknowledge D. Bolt, USDA (Beltsville, MD), for FSH. Pituitary hormones and FSH antisera were obtained through the National Hormone and Pituitary Program, NIDDK, NICHHD, and the USDA.

	Footnotes

 
¹ Approved for publication by the director, Oklahoma Agricultural Experimental Station. This work was supported in part by the National Research Initiative Competitive Grants Program Grants Office/USDA, Competition Grant 92–37203-8038, and NIH Grant HD-29845.

Received July 24, 1996.

	References

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