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J. Anim. Sci. 2003. 81:269-278
© 2003 American Society of Animal Science

Influence of estradiol, progesterone, and nutrition on concentrations of gonadotropins and GnRH receptors, and abundance of mRNA for GnRH receptors and gonadotropin subunits in pituitary glands of beef cows1,2

M. L. Looper*,3, J. A. Vizcarra*,2, R. P. Wettemann*,5, J. R. Malayer{dagger}, T. D. Braden§, R. D. Geisert* and G. L. Morgan{ddagger}

* Department of Animal Science, Oklahoma Agricultural Experiment Station; and {dagger} Departments of Physiological Sciences and and {ddagger} Medicine and Surgery, College of Veterinary Medicine, Oklahoma State University, Stillwater 74078-0425; and and § Department of Anatomy, Physiology, and Pharmacology, College of Veterinary Medicine, Auburn University, AL 36848-5520

5 Correspondence:
Phone: 405-744-6077; fax: 405-744-7390; E-mail:
rpw{at}okstate.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
Nutritionally induced anovulatory cows (n = 28) were used to determine the effect of steroids on regulation of synthesis and secretion of gonadotropins. Anovulatory cows were ovariectomized and received intravaginal inserts containing estradiol (E2), progesterone (P4), E2 and P4 (E2P4), or a sham intravaginal insert (C) for 7 d. Concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were quantified in serum and E2 and P4 were quantified in plasma. Cows were exsanguinated within 1 to 2 h after removal of intravaginal inserts and pituitary glands were collected and stored at -80°C until messenger ribonucleic acid (mRNA) for gonadotropin-releasing hormone receptor (GnRH-R) and gonadotropin subunits, pituitary content of GnRH-R, and LH and FSH were quantified. Pituitary glands from five proestrous cows were harvested to compare gonadotropin characteristics between ovariectomized, anovulatory cows and intact cows. Plasma concentrations of E2 were greater (P < 0.05) in E2-treated cows than in sham-treated cows. Concentrations of P4 were greater (P < 0.05) in cows treated with P4 than in sham-treated cows. Mean serum concentrations of LH and FSH were not significantly influenced by steroid treatments. However, frequency of LH pulses of ovariectomized, nutritionally induced anovulatory cows was increased (P < 0.05) by treatment with E2 and amplitude of LH pulses was greater (P < 0.05) in cows treated with E2 or P4 than in cows treated with E2P4 or sham-treated. Quantity of mRNA for LHß in the pituitary gland was greater when cows were treated with P4. Concentrations of LH in the pituitary gland were not affected by steroid treatments; however, pituitary concentrations of FSH were less (P < 0.1) in E2 cows than in sham-treated cows. The number of GnRH-R was increased (P < 0.05) in cows treated with E2, but P4 treatment did not influence the number of GnRH-R. Abundance of mRNA for GnRH-R, common {alpha}-subunit, and FSHß were not affected by treatments. Pituitary concentrations of LH were greater (P < 0.05) and concentrations of FSH were less (P < 0.05) in proestrous cows than in ovariectomized, anovulatory cows treated with or without steroids. Abundance of mRNA for GnRH-R, common {alpha}-subunit, LHß and FSHß were similar for proestrous and anovulatory cows. We conclude that treatment of nutritionally induced anovulatory cows with progesterone and estradiol may cause pulsatile secretion of LH.

Key Words: Beef Cows • Estradiol • Follicle-Stimulating Hormone • Luteinizing Hormone • Progesterone


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
Estradiol and progesterone regulate the synthesis and release of gonadotropins through negative and positive feedback effects on the central nervous system and the anterior pituitary (Kesner et al., 1981). Increased concentrations of estradiol and decreased progesterone in plasma are associated with the preovulatory LH surge in cattle (Wettemann et al., 1972; Chenault et al., 1975). Serum concentrations of LH initially decrease after estradiol treatment followed by a preovulatory-like LH surge in ovariectomized cows (Schoenemann et al., 1985). Estradiol influences neurotransmitters in the brain that control pulsatile secretion of GnRH (Smith and Jennes, 2001) and increases the sensitivity of the anterior pituitary gland to GnRH by increasing the number of GnRH receptors (GnRH-R) in cows (Schoenemann et al., 1985). Removal of steroids by gonadectomy increases gene expression of gonadotropin subunits (common {alpha}-subunit, LHß, and FSHß) in rats (Papavasiliou et al., 1986), sheep (Landefeld et al., 1984), and heifers (Roberson et al., 1992). The stimulatory effects of gonadectomy on messenger RNA (mRNA) for gonadotropin subunits is inhibited by estrogen treatment (Gharib et al., 1987; Herring et al., 1991). Reduced feed intake decreases concentration and pulse frequency of LH in cattle (Day et al., 1986; Richards et al., 1989; Bossis et al., 1999) and offers a model to determine the effects of estradiol and progesterone on secretion of LH.

Effects of physiological concentrations of steroids and nutrition on abundance of mRNA for gonadotropin synthesis have not been established in cows. Steroid treatment that stimulates follicular growth and ovulation in anestrous cows will increase reproductive efficiency. The hypotheses tested in this experiment were: 1) estradiol and progesterone alter concentrations of LH, FSH, and GnRH-R and abundance of mRNA for GnRH-R and gonadotropin subunits in pituitary glands of nutritionally induced anovulatory beef cows that are ovariectomized, and 2) nutritional deprivation alters the quantity of mRNA for GnRH-R and gonadotropin subunits in pituitary glands of beef cows.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Implications
 Literature Cited
 
Animal Model and Experimental Design
All animal procedures used in these studies were approved by the Oklahoma State University Institutional Animal Care and Use Committee. Twenty-eight multiparous, nonlactating, Angus x Hereford beef cows with normal estrous cycles (BW = 435 ± 10 kg) were fed a restricted diet of 2.7 kg of prairie hay (CP = 5.8%) and 35 g of a mineral mix (46.1% salt, 50.0% dicalcium phosphate, 0.4% copper sulfate, 0.5% zinc oxide and 3.0% mineral oil) daily to cause loss of 1% of BW/wk until they became anovulatory. Body weight and BCS (1 = emaciated, 9 = obese; Wagner et al., 1988) were evaluated every 2 wk; ovulation ceased when BCS averaged 3.8 ± 0.2. Blood samples were collected weekly via tail venipuncture in 10-mL tubes containing EDTA (0.1 mL of a 15% solution), placed on ice, and centrifuged (2,500 x g for 15 min) within 2 h. Plasma was decanted and stored at -20°C until progesterone was quantified. Cows were considered anovulatory when three consecutive plasma samples contained less than 1 ng/mL of progesterone.

Cows were ovariectomized and randomly assigned to treatment groups within 3 wk after the onset of anovulation. One week prior to the initiation of treatment (6 h after ovariectomy), cows were confined in individual stalls in a barn at 21 ± 4°C and 14 h of light. A polyvinyl jugular cannula (1.68 mm i.d., 2.39 mm o.d.; BB 317 v11, Bolab, Lake Havasu City, AZ) was inserted 2 d prior to treatment, to allow collection of frequent blood samples.

On d 0, cows received one of four treatments (n = 7/treatment): 1) an intravaginal insert containing progesterone (P4; EAZI-BREED CIDR, InterAg, Hamilton, New Zealand; 1.9 g of progesterone) designed to produce concentrations of progesterone similar to the early luteal phase of estrous cycles, 2) a sham intravaginal insert (silicone insert without progesterone) with 17ß-estradiol (E2; Sigma Chemical, St. Louis, MO) in a 60-mm silastic tube (3.35 mm i.d.; 4.65 mm o.d.; Dow Corning Co., Midland, MI) attached to the sham insert, designed to produce plasma concentrations of estradiol similar to the early luteal phase of the estrous cycle, 3) an intravaginal insert containing E2 and P4 (E2P4), or 4) a sham intravaginal insert (C).

Treatments were initiated on d 0 at 0800 h and continued through 0600 h on d 7. Blood samples were collected at 10-min intervals for 4 h 1 d prior (d -1) to treatments and at 10-min intervals for 8 h on d 6 of steroid treatment. Samples were allowed to clot for 24 h at 4°C and then centrifuged at 2,500 x g for 20 min. Serum was decanted and stored at -20°C until concentrations of LH and FSH were quantified. Daily blood samples were collected from d -1 to d 6 into 10-mL tubes containing EDTA, and plasma was decanted and stored at -20°C until progesterone and estradiol were quantified.

Five Angus x Hereford cows with a BCS of 5.0 and normal estrous cycles, were used to compare gonadotropin synthesis in cows during the proestrous phase of normal estrous cycles with cows that lost body weight and became anovulatory. Two days before exsanguination, cows that were between d 8 and 12 of the estrous cycle were treated with PGF2{alpha} (Lutalyse, 25 mg; Pharmacia & Upjohn, Kalamazoo, MI) to induce luteolysis and become proestrus. Ovaries were examined at exsanguination to ascertain that corpus luteum regression had occurred.

Pituitary Gland Collection
On d 7 at 0700 h, anovulatory cows were exsanguinated within 1 to 2 h after removal of intravaginal inserts. Intact cows with normal estrous cycles were exsanguinated 48 h after PGF2{alpha} treatment. Pituitary glands were removed, placed on ice, trimmed, sectioned midsagitally, and the posterior lobe was discarded. Anterior pituitary glands were weighed and frozen in liquid nitrogen (-72°C) within 40 min after exsanguination.

Hormone and Receptor Assays
For both LH and FSH assays, assay was a block in the statistical analysis, with all samples for the same number of cows per treatment quantified in each assay. Serum concentrations of LH were quantified by RIA as previously validated using antibody OSU-BLH-4-1 and NIH LH-B9 (National Hormone and Pituitary Program, Torrance, CA) as the standard (Bishop and Wettemann, 1993). Intra- and interassay coefficients of variation were 19 and 25%, respectively (n = 6 assays), with a sensitivity of 0.33 ng/mL. Dose-response curves for serum and anterior pituitary homogenates from cows were parallel to the standard curve. Concentrations of FSH in serum were determined by RIA as previously validated using antibody NIDDK-oFSH-I-1 with USDA-bFSH-I-2 as the standard (Vizcarra et al., 1997). Intra- and interassay coefficients of variation were 12 and 22%, respectively (n = 6 assays), with a sensitivity of 0.10 ng/mL. Dose-response curves for serum and anterior pituitary homogenates from cows were parallel to the standard curve.

Concentrations of LH and FSH in pituitary tissue were quantified by RIA. One-half of each pituitary gland was thawed, homogenized in buffer (10 mM Tris, 1 mM CaCl2, 0.25 M sucrose, pH 7.0) with three, 5-s bursts in a Tissue Tearor (Biospec, Bartlesville, OK). The crude homogenate was then homogenized in a ground-glass homogenizer (Tenbroeck Tissue Grinder, Kontes, Vineland, NJ), and then rehomogenized in a glass homogenizer (7-mL Dounce Tissue Grinder, Kontes). Tissue and buffer was maintained at 4°C. The homogenate was centrifuged at 16,000 x g for 15 min (4°C), and the supernatant was stored at -80°C until concentrations of LH and FSH were quantified. The precipitate was resuspended in assay buffer (10 mM Tris, 1 mM CaCl2, 0.3% [wt/vol] BSA, pH 7.0) to determine concentrations of GnRH-R.

Receptors for GnRH were quantified as described by Nett et al. (1987) with modifications (Vizcarra et al., 1997). Briefly, a standard curve was generated using several quantities of GnRH-R (0.32 to 16.8 fmol) from a pool of bovine pituitary membrane incubated with a constant quantity of [125I]buserelin ([D-Ser{tBu}6, Pro9 NHEt]GnRH, a gift from Hoescht Roussel Pharmaceuticals, Summerville, NJ; 4.8 fmol). The [125I]buserelin is typically 40 to 50% bindable to excess receptor with a specific activity estimated by self-displacement to be 1,100 Ci/nM. 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 (Scatchard, 1949). Binding of [125I]buserelin to sample tissues was directly compared to the standard curve to determine the number of GnRH-R in sample tissues. Steady state binding of [125I]buserelin was attained by 2 h at 4°C and was maintained for at least 12 h. Receptor assay results are expressed in femtomoles per mg protein.

Concentrations of progesterone in plasma were determined by solid-phase RIA (Coat-A-Count progesterone kit, Diagnostic Products Corp., Los Angeles, CA; Vizcarra et al., 1997). Intraassay coefficient of variation was 4% (n = 1 assay). Concentrations of estradiol-17ß were quantified by RIA (Estradiol MAIA assay kit, Polymedics, New York, NY) with modifications (Vizcarra et al., 1997). Intraassay coefficient of variation was 9% (n = 1 assay).

Analyses of Messenger Ribonucleic Acid
Total RNA was isolated from pituitary tissue (0.5 g) by homogenization in 5 mL of TRIZOL reagent (Life Technologies, Inc., Gaithersburg, MD) in polypropylene tubes (Corning #25319-15, Corning, NY) on ice with a VirTishear homogenizer (Gardiner, NY). Homogenates were incubated at room temperature for 5 min. Chloroform (Molecular Biology Grade; Fisher Scientific, Pittsburg, PA) was added (1 mL) to each sample and mixed for 15 sec. After centrifugation (5,000 x g for 30 min at 4°C), the aqueous phase was transferred to a new polypropylene tube and the RNA was precipitated with 2.5 mL of isopropyl alcohol (99.9% purity; EM Science, Gibbstown, NJ). Samples of RNA were incubated in polypropylene tubes at room temperature for 10 min and centrifuged at 5,000 x g for 25 min at 4°C. The supernate was removed and the RNA pellet was washed with 5 mL of 75% ethanol and centrifuged (5,000 x g for 10 min at 4°C). The RNA pellet was dissolved in 200 µL of TE buffer (10 mM Tris-Cl, 1 mM EDTA, pH 7.4) and quantified by spectrophotometry at 260 nm. The purity of RNA was determined from the 260/280 nm of absorbance. The extraction procedure yielded 260/280 nm absorbance ratios of 1.6 to 1.7.

Integrity of mRNA for specific genes of interest was determined by agarose gel electrophoresis and Northern analysis of total RNA from one control and one steroid-treated (E2P4) cow. After being transferred to nylon membranes (ICN, Biotrans Nylon Membranes, Irvine, CA), RNA was hybridized to radiolabeled ovine GnRH-R (Turzillo et al., 1994), bovine common {alpha}-subunit (Erwin et al., 1983), bovine LHß (Maurer, 1985), or bovine FSHß (Kim et al., 1988) complementary DNA (cDNA). Each cDNA probe was radiolabeled with 32P using the random hexamer priming method (Prime-a-Gene System, Promega Corp., Madison, WI). Three GnRH-R mRNA transcripts were evident on Northern blots at 5.2, 3.5 and 2.0 kilobases (data not presented). This finding is in agreement with Vizcarra et al. (1997). Single transcripts were observed for common {alpha}-, LHß- and FSHß-subunit mRNA.

Concentrations of pituitary mRNA were determined by slot blot analyses. Thirty-five and 17.5 µg of total RNA from each sample were applied in duplicate to nylon membranes (ICN, Biotrans Nylon Membranes). Membranes were baked at 80°C for 2 h to crosslink RNA to the membrane. Slot blot membranes were hybridized at 42°C for 16 to 18 h to radiolabeled 32p cDNA encoding ovine GnRH-R, bovine {alpha}-, LHß-, and FSHß-subunits prepared as previously described. Hybridization buffer consisted of 0.5 M Na2PO4, 7% SDS, 1% BSA, 1mM EDTA, and 0.1 mg/mL denatured salmon sperm DNA (Church and Gilbert, 1984). Membranes were washed in 0.5 x standard sodium citrate (SSC) at 65°C for {alpha}-subunit, 0.5 x SSC at 42°C for FSHß-subunit, and 1 x SSC at 42°C for LHß-subunit and GnRH-R. To adjust for differences in RNA loading among samples, membranes were stripped of GnRH-R and subunit probes by washing in 0.2 M NaOH at room temperature for 2 h and reprobed with radiolabeled cDNA probe complementary to 18S ribosomal RNA (pTRI RNA 18S, Ambion, Inc., Austin, TX). Membranes were hybridized with the 18S ribosomal RNA probe at 42°C for 16 to 18 h and washed with 0.1 x SSC at 65°C. Membranes were exposed to film (Kodak, Rochester, NY) for 0.5 to 5 d at -80°C and developed. Autoradiographs were quantified with a scanning densitometer and image analysis software (Image 1.60, NIH, Washington, DC). Results are expressed as arbitrary densitometric units. Amounts of radiolabeled 18S hybridized to membranes for each sample did not differ (P > 0.10) indicating similar amounts of RNA were loaded.

Pulse Analyses
Pulse frequency and amplitude of LH and FSH in serum samples were determined using the pulsar program (Merriam and Wachter, 1982). To determine if variations in hormone concentrations in serial samples are pulses in hormone secretion or just random variations in concentrations, appropriate G values were chosen. The value for G1 is usually set at 99 to prevent identifying a one-sample increase in concentration as a pulse, which by our definition of pulses cannot occur due to the frequency at which samples were collected. The G5 value was also set at 99 to avoid the false positive determination of a small increase, followed by a return to baseline concentration, as a pulse. The G values for both LH and FSH were G1 = 99, G2 = 4.5, G3 = 4, G4 = 3.5 and G5 = 99.

Statistical Analyses
Cows treated with and without P4, and cows treated with and without E2 were used in a 2 x 2 factorial arrangement. Analyses of variance were performed using the GLM procedure of SAS (SAS Inst., Inc., Cary, NC) to determine the effects of treatment with progesterone or estradiol on concentrations and content of LH and FSH in the pituitary gland, pulse frequency, and amplitude of LH and FSH in serum, concentration of GnRH-R, and amounts of mRNA for GnRH-R, {alpha}-subunit, LHß and FSHß. Effects of steroid treatment on serum concentrations of LH and FSH, and concentrations of progesterone and estradiol in daily plasma samples, were determined by the MIXED procedure of SAS with the model including effects of assay (block), estradiol, progesterone, sample and the interactions. Treatment means were compared using the PDIFF statement of SAS when protected by a significant (P < 0.05) treatment effect. Analyses of variance were performed using the GLM procedure of SAS to determine differences between proestrous cows and anovulatory cows treated with steroids and, when treatment was significant, means were compared with Scheffe’s multiple comparisons test (Steel and Torrie, 1980).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Implications
 Literature Cited
 
Steroid Treatment of Anovulatory Cows
Plasma concentrations of estradiol were greater (P < 0.01) during the 6 d after treatment of ovariectomized, anovulatory cows with E2 or E2P4 compared with pretreatment (d 0) concentrations (Table 1Go) or concentrations during the 6 d in cows not treated with E2 (data not shown). Concentrations of progesterone were greater (P < 0.01) during the 6 d after treatment with P4 or E2P4 compared with pretreatment (d 0) concentrations (Table 1Go) or concentrations in cows not treated with P4 (data not shown).


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  		Table 1. Least squares mean concentrations of estradiol and progesterone in plasma of ovariectomized, anovulatory cows treated with intravaginal inserts containing estradiol and/or progesterone
 
Mean serum concentrations of LH and FSH on d -1 did not differ among treatments (P > 0.1) and averaged 1.82 ± 0.06 and 0.58 ± 0.01 ng/mL, respectively (Table 2Go and 3Go). Treatments did not influence (P > 0.1) mean concentrations of LH in serum of cows on d 6 (2.08 ± 0.04 ng/mL; Table 2Go). Similarly, mean FSH concentrations were not affected (P > 0.1) by steroid treatments for 6 d (0.55 ± 0.02 ng/mL; Table 3Go).


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  		Table 2. Least squares mean concentration, pulse frequency, and pulse amplitude of luteinizing hormone in serum on d -1 and 6 in ovariectomized, anovulatory cows treated with intravaginal inserts containing estradiol (E2), progesterone (P4), E2 and P4 (E2P4), or with a sham intravaginal insert (C) for 7 d
 

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  		Table 3. Least squares mean concentration, pulse frequency, and pulse amplitude of follicle-stimulating hormone in serum on d -1 and 6 in ovariectomized, anovulatory cows treated with intravaginal inserts containing estradiol (E2), progesterone (P4), E2 and P4 (E2P4), or with a sham intravaginal device (C) for 7 d
 
Frequencies of LH and FSH pulses in serum on d -1 averaged 1.7 ± 1.1 and 1.4 ± 1.0 pulses/4 h, respectively, and were not influenced by treatment (Table 2Go and 3Go). Cows treated with E2 for 6 d had increased (P < 0.05) LH pulse frequency compared with cows not treated with E2. Frequency of FSH pulses was not influenced by treatment (Table 3Go).

Amplitude of LH and FSH pulses in serum on d -1 averaged 1.01 ± 0.17 and 0.22 ± 0.02 ng/mL, respectively, and were not influenced by treatment (Table 2Go and 3Go). There was an E2 x P4 effect (P < 0.05) on amplitude of LH pulses. Cows treated with E2 or with P4 had greater (P < 0.05) amplitudes of LH pulses on d 6 than sham and E2P4 cows. Amplitudes of FSH pulses were not influenced by treatments (Table 3Go).

Weight of pituitary glands (1.86 ± 0.07 g) and concentrations of LH in pituitary glands of anovulatory cows were not affected (P > 0.1) by treatment with E2 and P4. However, FSH concentrations in pituitary glands of cows treated with E2 tended to be reduced (P = 0.08) compared with sham-treated cows or cows treated with P4 alone (Figure 1Go). Content of LH and FSH in the pituitary gland was not influenced by treatment.



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  		Figure 1. Least square mean concentrations of LH and FSH in pituitary glands of proestrous cows (I) and ovariectomized, anovulatory cows treated with intravaginal inserts containing estradiol (E2), progesterone (P4), E2 and P4 (E2P4) or a sham intravaginal insert (C) for 7 d. a,bDifferent letters above bars indicate that, within a hormone, means differ among anovulatory cows (P < 0.1; LH mean square error = 0.061; FSH mean square error = 0.002). y,zDifferent letters, above bars indicate that, within hormone, means differ between proestrous and anovulatory cows (P < 0.001; LH mean square error = 0.237; FSH mean square error = 0.002).

 
Concentrations of GnRH-R in pituitary glands were greater (P < 0.05) in cows treated with E2 or E2P4 compared with P4-treated or sham cows (Figure 2Go). Concentrations of GnRH-R mRNA in the pituitary gland were not influenced (P > 0.1) by steroid treatments (Figure 2Go).



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  		Figure 2. Least square mean concentrations of GnRH receptors (GnRH-R) and GnRH-R messenger RNA (mRNA) in pituitary glands of proestrous cows (I) and ovariectomized, anovulatory cows treated with intravaginal inserts containing estradiol (E2), progesterone (P4), E2 and P4 (E2P4), or a sham intravaginal insert (C) for 7 d. a,bDifferent letters above bars indicate that, within GnRH-R characteristic, means differ among anovulatory cows (P < 0.05; GnRH-R mean square error = 75.5; GnRH-R mRNA mean square error = 162.7).

 
Amounts of common {alpha}-subunit and FSHß mRNA in the pituitary were not influenced (P > 0.1) by treatment (Figure 3Go). Concentrations of LHß mRNA were increased (P < 0.05) in cows treated with P4 compared with cows not treated with P4 (Figure 3Go).



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  		Figure 3. Least square mean concentrations of common {alpha}-, LHß-, and FSHß-subunit messenger RNA (mRNA) in pituitary glands of proestrous cows (I) and ovariectomized, anovulatory cows treated with intravaginal inserts containing estradiol (E2), progesterone (P4), E2 and P4 (E2P4), or a sham intravaginal insert (C) for 7 d. a,bDifferent letters above bars indicate that, within gonadotropin subunit, means differ among anovulatory cows (P < 0.05; {alpha}-subunit mean square error = 7.3; LHß mean square error = 622.2; FSHß mean square error = 17.1).

 
Proestrous Cows vs Anovulatory Cows With or Without Steroids
Weights of anterior pituitary glands were similar (P > 0.1) for proestrous cows (1.64 ± 0.14 g) and ovariectomized, anovulatory cows with or without steroids (1.86 ± 0.07 g). Concentration of LH in the pituitary gland was greater (P < 0.05) and concentration of FSH was less (P < 0.05) in proestrous cows than in ovariectomized, anovulatory cows (Figure 1Go). Similarly, content of LH in the pituitary gland was greater (P < 0.05), and pituitary content of FSH was less (P < 0.05) in proestrous cows than in ovariectomized, anovulatory cows.

Abundance of mRNA for GnRH-R, common {alpha}-, LHß- and FSHß-subunits did not differ (P > 0.1) between proestrous cows and ovariectomized, anovulatory cows with or without steroid treatment (Figure 2Go and 3Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Implications
 Literature Cited
 
Treatment of ovariectomized, nutritionally induced anovulatory cows with an intravaginal insert containing P4 for 6 d increased progesterone in plasma similar to concentrations observed during the early luteal phase of estrous cycles (Stabenfeldt et al., 1969; Wettemann et al., 1972). Concentrations of estradiol in plasma were increased in cows receiving an intravaginal insert containing E2 and concentrations were similar to values during the luteal phase of estrous cycles (Wettemann et al., 1972; Glencross et al., 1973; Bossis et al., 1999).

Treatment of ovariectomized, nutritionally induced anovulatory cows with E2, P4, or E2P4 for 6 d did not influence mean concentrations of LH and FSH in serum. Removal of the regulatory control of the ovary increases concentrations of LH (Schallenberger and Peterson, 1982; Anderson et al., 1985) and FSH in serum (Ireland et al., 1983) in cattle with normal estrous cycles. Concentrations of LH in serum are reduced in anestrous cows (Richards et al., 1989; Vizcarra et al., 1997) and feed-restricted heifers (Kurz et al., 1990; Bossis et al., 1999). Richards et al. (1991) found that concentrations of LH in serum were not influenced by estradiol treatment during the first 10 d after ovariectomy in nutritionally anestrous cows. Similarly, steroid treatment for 6 d may not be sufficient to influence mean concentrations of LH in anovulatory beef cows.

The number of pulses of LH in serum was greater in cows treated with E2 for 6 d than in control or P4-treated cows. Steroids act at the hypothalamus, anterior pituitary gland or at both sites to regulate gonadotropin release (Goodman and Karsch, 1980; Rahe et al., 1980). Treatment of anovulatory cows with E2 for 6 d may stimulate GnRH release, increasing pulsatile release of LH. Estradiol induced a preovulatory-like surge of GnRH in the cerebrospinal fluid of ovariectomized cows, which was associated with a LH surge (Gazal et al., 1998). Increased pulse frequency of LH in anovulatory cows treated with E2 also may be due to the effects of estrogens on the anterior pituitary gland. In vitro treatment of rat (Drouin et al., 1976) and cow (Padmanabhan et al., 1978) pituitary cells with estradiol increased LH release and synthesis. Estradiol also increases the sensitivity of the anterior pituitary gland to GnRH in cows (Kesner et al., 1981). The number of GnRH-R in the anterior pituitary gland of cows is increased after exogenous estrogen (Schoenemann et al., 1985).

Pulse amplitude of LH was greater in cows treated with P4 or E2 than controls or cows treated with P4 and E2. Progesterone reduces frequency of LH pulses and increases amplitude of pulses in cattle (Rahe et al., 1980) and sheep (Goodman and Karsch, 1980). Progesterone treatment decreases GnRH secretion in sheep (Karsch et al., 1987). Increased pulse amplitude of LH is probably a result of E2 increasing GnRH secretion and/or increasing the number of GnRH-R in the anterior pituitary gland. Cows fed restricted diets released more LH in response to exogenous GnRH than did moderate-condition cows (Beal et al., 1978; Rasby et al., 1991). Increased pulse amplitude of LH in anovulatory cows treated with P4 or E2 indicates that the pituitary gland is not depleted of gonadotropins in underfed cattle. The presence of either steroid alone, but not the combination of both P4 and E2 in the current study, increased the amplitude of LH pulses.

Steroid treatment for 7 d did not influence concentration or content of LH in the anterior pituitary gland of anovulatory cows, but cows treated with E2 had reduced pituitary FSH concentrations. Duration and amount of steroid treatment may affect pituitary concentrations of LH and FSH. Treatment of ovariectomized cows with one injection of estradiol (1 mg) increased pituitary gland concentrations of LH and FSH 20 h after estradiol (Schoenemann et al., 1985); however, the reproductive state of the cows before ovariectomy was not reported. Steroids may influence LH and FSH synthesis and release differently. For example, estradiol treatment of ovariectomized cows caused a LH surge without altering serum concentrations of FSH (Schoenemann et al., 1985).

Concentrations of GnRH-R increased when anovulatory cows were treated with E2 for 7 d. Estradiol also increased concentrations of GnRH-R in the anterior pituitary gland of cattle with normal estrous cycles (Kaltenbach et al., 1974; Kesner et al., 1981; Schoenemann et al., 1985) and the stimulatory effect of estradiol on number of GnRH-R was not inhibited by progesterone in wethers (Sakurai et al., 1997). When ewes were treated with estradiol there was a 2.5-fold increase in the number of GnRH-R in the pituitary (Gregg and Nett, 1989). Receptors for GnRH can be influenced by GnRH secretion (Braden and Conn, 1991). Estradiol and GnRH had synergistic effects on ovariectomized ewes in which the pituitary gland was disconnected from the hypothalamus, resulting in an increased number of GnRH-R and increased GnRH-R mRNA (Kirkpatrick et al., 1998). Thus, estradiol may increase GnRH-R by increasing GnRH release from the hypothalamus and/or by directly influencing GnRH-R at the anterior pituitary gland.

Concentrations of GnRH-R mRNA were not influenced by steroid treatment for 7 d. Concentrations of GnRH-R mRNA are increased during the preovulatory period in sheep (Brooks et al., 1993; Turzillo et al., 1994) and rats (Bauer-Dantoin et al., 1993). Duration of exposure to steroids influences amounts of mRNA for GnRH-R. Acute treatment of ovariectomized ewes (Hamernik et al., 1995; Turzillo et al., 1995) and wethers (Adams et al., 1996; 1997) with estradiol increased GnRH-R mRNA. However, chronic treatment with estradiol for 7 d decreased GnRH-R mRNA in ovariectomized rats (Kaiser et al., 1993). Chronic exposure (7 to 8 d) to progesterone reduced concentrations of GnRH-R mRNA in wethers (Sakurai et al., 1997). Pituitary concentrations of GnRH-R mRNA increased 12 h after PGF2{alpha} induced luteolysis in intact ewes (Turzillo et al., 1994). This increase in mRNA for GnRH-R occurred at a time when concentrations of progesterone were decreased and prior to significant increases in concentrations of estradiol, indicating that progesterone may mediate pretranslation of GnRH-R (Turzillo et al., 1994). In addition, the stimulatory effect of estradiol for gene expression for GnRH-R is prevented during the luteal phase in ewes, but the inhibition may not be due to progesterone (Turzillo et al., 1998).

Concentrations of common {alpha}- and FSHß-subunit mRNA were not influenced by steroid treatments. However, pituitary concentrations of LHß mRNA were greater in cows treated with P4 than in E2-treated and control cows. Progesterone inhibits LH release in cattle (Walters et al., 1982) and sheep (Moss et al., 1981). Withdrawal of exogenous progesterone increases LH secretion in postpartum anestrous cows (Garcia-Winder et al., 1987) and pubertal heifers (Anderson et al., 1996). Progesterone treatment for 7 d increased the synthesis of LHß mRNA without altering mean concentration or pulse frequency of LH in the current study. Similarly, steady-state concentrations of common {alpha}-subunit and LHß mRNA are uncoupled from pulsatile LH release in the sexually maturing heifer (Roberson et al., 1992). Duration of steroid exposure also may influence amounts of mRNA for gonadotropins. Acute exposure of ovariectomized ewes to estradiol (12 h) decreased concentrations of mRNA for all three gonadotropin subunits followed by increases in common {alpha}- and LHß mRNA subunits after 24 h, with no affect on FSHß mRNA (Herring et al., 1991). Further exposure to estradiol decreased concentrations of common {alpha}-subunit and LHß mRNA after 4 d, whereas concentrations of FSHß subunit mRNA were decreased after 8 d (Herring et al., 1991).

Proestrous cows had a greater concentration and content of LH in the pituitary gland than ovariectomized, anovulatory cows with or without steroids. Concentrations and content of LH in the pituitary gland of anovulatory cows were 25 and 27%, respectively, the concentrations and content in the pituitary gland of proestrous cows. Concentrations and content of LH in the current study are similar to those observed during proestrus in beef cows (Funston et al., 1995) and dairy cows (Hackett and Hafs, 1969) prior to LH release at ovulation. Pituitary concentrations of LH and FSH were increased 20 h after estradiol treatment of ovariectomized cows (Schoenemann et al., 1985).

Concentrations and content of FSH in the pituitary gland were reduced in proestrous cows compared with ovariectomized, anovulatory cows with or without steroids. Concentrations and content of FSH in the pituitary gland of proestrous cows were decreased 65 and 70%, respectively, compared with anovulatory cows. Concentrations and content of FSH are less than those reported for proestrous dairy cows (Hackett and Hafs, 1969). These differences could be due to cows in different stages of estrus or method of quantifying FSH. Increased pituitary content of FSH in ovariectomized cows is likely due to removal of ovarian hormones (inhibin and estradiol) that regulate FSH (Ireland et al., 1983; Robertson et al., 1985; 1988).

Abundance of mRNA for GnRH-R, common {alpha}-subunit, LHß, and FSHß were similar for proestrous cows and ovariectomized, anovulatory cows. Increased steady-state concentrations of GnRH-R mRNA during the preovulatory period are common in rats (Bauer-Dantoin et al., 1993) and sheep (Brooks et al., 1993; Turzillo et al., 1994). Acute exposure to estradiol increases common {alpha}-subunit and LHß mRNA in ovariectomized ewes (Herring et al., 1991). Treatment with progesterone had no affect on pituitary concentrations of mRNA encoding for common {alpha}-, LHß-, and FSHß-subunits in ovariectomized sheep (Hamernik et al., 1987). Differences in animal model (intact vs castrated) as well as the physiological state of the animal at the time of pituitary harvest may account for differences in studies of steady-state amounts of mRNA. Winters (1996) found a relationship between plasma concentrations of FSH and FSHß mRNA in intact rats, but not in castrated rats. Discrepancies when comparing amounts of mRNA between studies may be due to slight differences in the time at which pituitary glands were collected. Alexander and Miller (1982) proposed the half-life of the common {alpha}-subunit was 51 h, whereas the ß-subunit is thought to be less stable with a half-life of 12 to 16 h (Hall and Miller, 1986; Hamernik and Nett, 1988; Di Gregorio and Nett, 1995).

In summary, estradiol treatment of ovariectomized, nutritionally induced anovulatory cows increased the frequency of LH pulses in serum and treatment with either E2 or P4 (but not both) increases the amplitude of LH pulses. The amount of GnRH receptors in the anterior pituitary gland, but not the amount of mRNA for GnRH-R, was increased by treatment with E2. Treatment with E2 decreased concentrations of FSH in the anterior pituitary gland. Progesterone treatment increased the abundance of mRNA for LHß in the anterior pituitary gland, and mRNA for FSHß was not influenced by either E2 or P4 treatment. Abundance of mRNA for GnRH receptor and gonadotropin subunits was not different for proestrus cows compared with nutritionally induced anovulatory cows with or without treatment with steroids. Concentrations of LH were greater and concentrations of FSH were less in the anterior pituitary of proestrus cows compared with ovariectomized, nutritionally induced anovulatory cows with or without treatment with E2 and P4.


   Implications
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
Literature Cited
 
Resumption of estrus and luteal activity by 85 d after calving is necessary to maintain a 12-mo calving interval. Treatment of anestrous cows with concentrations of estradiol similar to those during the luteal phase may increase luteinizing hormone pulse frequency and gonadotropin-releasing hormone receptors in the pituitary gland, which are necessary for initiation of estrous cycles. Exposure to concentrations of progesterone in plasma similar to those during the estrous cycle could increase synthesis of luteinizing hormone ß messenger ribonucleic acid in the anterior pituitary gland of anovulatory beef cows. Increased synthesis of luteinizing hormone ß could increase pituitary content of luteinizing hormone, and cause a luteinizing hormone surge and ovulation to occur sooner after calving.


   Footnotes
 
1 Approved for publication by the Director, Oklahoma Agric. Exp. Stn. This research was supported under project H-2331 and in part by Oklahoma Center for the Advancement of Science and Technology (HR4-031). Back

2 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 USDA. We thank T. M. Nett (Colorado State University) for ovine GnRH receptor cDNA and R. A. Maurer (Oregon Health Science University) for bovine common {alpha}-subunit, LHß, and FSHß cDNA. Appreciation is expressed to J. F. McAllister, Pharmacia & Upjohn for donation of Lutalyse. Technical assistance of M. Anderson, S. Welty, L. Mackey, R. Jones, C. Lents, and K. Vonnahme is appreciated. Back

3 Current address: USDA-ARS, Dale Bumpers Small Farms Research Center, Booneville, AR 72927. Back

4 Current address: Department of Animal Sciences and Food Technology, Texas Tech University, Lubbock 78409-2141. Back

Received for publication March 19, 2002. Accepted for publication September 10, 2002.


   Literature Cited
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 


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