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Biology of Reproduction 59, 124-130 (1998)
©Copyright 1998 Society for the Study of Reproduction, Inc.

Estradiol Increases Relative Amounts of Insulin-Like Growth Factor Binding Protein (IGFBP)-3 in Serum and Expression of IGFBP-2 in Anterior Pituitaries of Ewes1

J.A. Clapper3,a, J.L. Snydera, A.J. Robertsb, D.L. Hamernik4,c, , and G.E. Moss2,a

a University of Wyoming, Department of Animal Science, Laramie, b Wyoming 82071 USDA, Agricultural Research Service, R.L. Hruska U.S. Meat Animal Research Center, Clay Center, Nebraska 68933 c University of Arizona, Department of Physiology, Tucson, Arizona 85718


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study determined whether estradiol regulates insulin-like growth factor (IGF)-I and IGF binding proteins (IGFBPs) in the pituitary gland, hypophyseal stalk median eminence (SME), and circulation concomitantly with effects on LH. Ovariectomized ewes received an estradiol implant or no implant during the anestrous season and were slaughtered 80 days later. Estradiol suppressed serum LH to a greater extent during anestrus than after onset of the breeding season (Days 60 and 75). Amounts of mRNA for LHß subunit were decreased by estradiol, but mRNA for {alpha} and FSHß subunits were not affected. Estradiol increased serum IGF-I, IGFBP-3, and IGFBP-4 throughout the treatment period, but it did not influence other IGFBPs in serum. In response to estradiol, pituitary IGFBP-2 tended to increase and mRNA for IGFBP-2 increased twofold. Other IGFBPs in the pituitary gland were not influenced by estradiol. In the SME, IGFBP-2, IGFBP-5, and the 40-kDa IGFBP-3 were increased by estradiol. Thus, estradiol influences both the IGF and gonadotropin systems in sheep. Estradiol influences on gonadotroph function may be mediated by alterations in the IGF system.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Insulin-like growth factor (IGF)-I is a protein produced and secreted from the liver in response to growth hormone secretion from the anterior pituitary gland [1]. It is normally bound to one of at least six IGF binding proteins (IGFBPs) that vary in molecular weight, tissue distribution, and affinity for IGF-I [2]. Many extrahepatic tissues also express IGF-I (reviewed in [3]). Because IGF-I is produced by multiple tissues and because receptors for IGF-I are widespread throughout the body including neuronal tissue [2], IGF-I is thought to function through paracrine and autocrine mechanisms as well as through an endocrine mechanism [4, 5].

IGF-I and IGFBPs are produced in the anterior pituitary gland and brain [6-10]. In addition, high concentrations of IGF-I are present in the cerebral cortex and hypothalamus of rats [11]. Roles for the IGF system in mediating gonadal [12, 13] and uterine function [14] have been reviewed, and potential actions of IGF-I in central control of reproduction are emerging. In vitro studies demonstrated that insulin-like peptides participate in the control of glucose transporters in the brain [15], and regulate expression [16] and release of GnRH [17] and LH [18–20].

Additional evidence indicates that estrogen regulates expression of the IGF system [3, 21]. Estradiol increased the production of IGF-I and IGFBP-3 in pituitary tumor cells [22] and increased IGFBP-2 expression in the anterior pituitary gland [23] of rats. The pituitary gland undergoes a well-described hypertrophy and hyperplasia in response to estrogens [24, 25]. Zeranol, a synthetic estrogen used to stimulate growth in domestic animals, increased serum concentrations of IGF-I and weight of the anterior pituitary gland in lambs [26].

IGF-I and IGFBPs were identified in ovine [27–29] and bovine [30] anterior pituitary glands. Furthermore, relative amounts of pituitary IGFBPs changed throughout the bovine estrous cycle [30]. The current study was conducted to determine whether estradiol regulates components of the IGF system in the ovine anterior pituitary gland and the hypophyseal stalk median eminence (SME) concomitantly with decreased secretion of LH.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mature, chronically ovariectomized Western range ewes were randomly assigned to receive s.c. a 10-mm Silastic implant containing crystalline estradiol-17ß (Sigma, St. Louis, MO; E; n = 6) or no implant (C; n = 4) on July 1 (Day 1). These implants were designed to maintain basal circulating concentrations of estradiol near 5 pg/ml [31–33]. This treatment inhibits the secretion of LH during the anestrous season but not during the breeding season [33, 34]. Ewes were fed dehydrated alfalfa pellets at 100% National Research Council requirements for net energy maintenance and had ad libitum access to water and trace mineral salt blocks. All experimental procedures were approved by the University of Wyoming Animal Care and Use Committee.

A single blood sample was obtained by jugular venipuncture from all ewes one day before they received estradiol implants (Day 0) to confirm initial similarity in circulating concentrations of IGF-I and IGFBPs among ewes. Subsequently, blood samples were collected before feeding at 12-min intervals for 6 h on Days 7, 30, 60, and 75. Days 60 and 75 were near (September 1) or after (September 15) the onset of the breeding season in comparable intact ewes maintained at the University of Wyoming. Blood was allowed to clot overnight at 4°C; then serum was separated by centrifugation (1500 x g; 30 min) and stored at -20°C until assayed.

Ewes were slaughtered on Day 80, and pituitary glands and SMEs were collected as described previously [35]. Pituitary glands were trimmed of connective tissue and bisected midsagittally. Tissues were wrapped in foil, frozen in liquid nitrogen, and stored at -70°C.

Serum concentrations of LH were determined in duplicate by RIA [36] in all samples collected at 12-min intervals on Days 7 through 75, using the antisera described by Adams et al. [37] at an initial dilution of 1:100 000. Radioiodinated LH (LER 1268–1; NIDDK) was used as the labeled antigen, and concentrations of LH were expressed as nanograms of NIH-LH-S18. Sensitivity of the assay was 0.4 ng/ml. Intraassay and interassay CV were 13.0% and 12.3%, respectively (n = 3 assays). Concentrations of LH in anterior pituitary gland homogenates (diluted 1:10 000 to 1:100 000 in PBS:0.1% gelatin) were measured in a single assay with an intraassay CV of 4.7%.

Pituitary concentrations of FSH were determined as described by Bolt [38] using ovine FSH (NIDDK-oFSH-I-S1AFP-21) as the radioiodinated antigen, ovine FSH (NIDDK-oFSH-RP-1) as the standard, and NIDDK-anti-oFSH–1 at an initial dilution of 1:80 000. Concentrations of FSH in anterior pituitary homogenates (1:1000 and 1:10 000 dilution in PBS–0.1% gelatin) were determined in a single assay with an intraassay CV of 5.5%.

Serum concentrations of IGF-I were determined in duplicate as described previously [29, 39] only in the first sample collected on Days 0 through 75 because serum concentrations of IGF-I are relatively constant throughout the day [40]. IGFBPs were extracted from serum [41] using a 1:16 ratio of serum to acidified ethanol (12.5% 2 N HCL:87.5% absolute ethanol). Recombinant human IGF-I (DRG010; Bachem, Torrance, CA) was used as the radioiodinated antigen and standard. Antisera UB3-189 (National Hormone and Pituitary Program, NIDDK) was used at an initial dilution of 1:2000. Sensitivity of the assay was 1.95 ng/ml. Intraassay and interassay CV were 10.2% and 15.1%, respectively. Concentrations of IGF-I in the anterior pituitary gland and SME were measured in a single assay [29] with an intraassay CV of 4.6%.

Relative amounts of IGFBPs in tissue and in the serum samples collected on Days 0 through 75 were analyzed by one-dimensional SDS-PAGE [42] and ligand blotting [43, 44] as described previously [29]. Briefly, serum (5 µl) or aliquots of homogenized tissue samples (50 µg and 75 µg total protein equivalents for pituitary and SME, respectively) were electrophoresed through a 4% stacking gel and a 10% separating gel. Proteins were electrophoretically transferred to nitrocellulose membranes (NitroBind, 0.22 µm, #EP2HY00010; Micron Separations, Inc., Westboro, MA), and IGFBP activity was detected by incubating membranes with 125I-IGF-I (1 000 000 cpm/ml Tris-buffered saline, 1% BSA [#A-7030; Sigma Chemical Co.], 0.1% Tween-20). Membranes were then exposed to autoradiographic film, and banding intensity was quantified by an LKB Bromma Ultrascan Laser Densitometer (Pharmacia LKB, Uppsala, Sweden).

The identity of IGFBP-2, -4, and -5 in serum and tissues was confirmed by immunoprecipitation of proteins in selected samples with polyclonal antibodies (06-107; 06-109; 06-110, respectively; Upstate Biotechnologies, Inc., Lake Placid, NY). The resulting supernatants, precipitates, and a nonimmunoprecipitated serum sample were subjected to ligand blot analysis as described previously [45]. On the basis of the similarity of kDas of IGFBPs identified in cattle [30], the IGFBP detected as a 40/44-kDa doublet in serum and a 36/40 kDa doublet in tissues was presumed to be IGFBP-3.

Northern and Dot Blots

Total RNA was extracted from half of each anterior pituitary gland using TriReagent (Molecular Research Company, Cincinnati, OH). Purity of RNA was determined by measuring the A260/A280 ratio. The ratio of all samples ranged from 1.7 to 2.0. The small size of the SME tissues (i.e., 30–50 mg) precluded extraction of RNA.

Relative amounts of mRNA for {alpha}, LHß, and FSHß subunits were determined by dot blot analysis and hybridization with radioactive cDNA probes encoding bovine {alpha} subunit [46], LHß subunit [47], or FSHß subunit [48]. Hybridization of total cellular RNA with a radioactive cDNA probe encoding rat cyclophilin [49] was conducted to control for unequal loading of RNA. Hybridization and wash conditions were similar to those described previously [50]. After hybridization, relative amounts of mRNA for each of the subunits (expressed as arbitrary densitometric units; ADU) were determined by phosphoimagery (Molecular Dynamics, Sunnyvale, CA). After confirming that estradiol treatment did not affect relative amounts of cyclophilin mRNA, relative abundance of gonadotropin subunit mRNA in individual pituitaries was normalized to relative amounts of cyclophilin mRNA (ADU gonadotropin/ADU cyclophilin) to account for unequal loading of RNA.

Northern blots for IGFBP-2 were performed on 10 µg of total anterior pituitary RNA according to the method of Sambrook and coworkers [51], and RNA was transferred to nylon membranes (BioTrans; ICN, Irvine, CA). Membranes were prehybridized in a solution containing 50% deionized formamide, 250 µg/ml boiled salmon sperm DNA, single-strength Denhardt's solution, 50 µg/ml Poly A RNA, 0.1% SDS, and 5-strength SSC (single-strength SSC is 0.15 M sodium chloride, 0.015 M sodium citrate) for 2 h at 65°C. Radioactive cRNA probes encoding 397 basepairs (bp) of rat IGFBP-2, [52] and 743 bp of rat cyclophilin [49] were prepared by labeling with [{alpha}-32P]uridine triphosphate (UTP; 10 mCi/ml; Amersham, Arlington Heights, IL). Blots were probed only for IGFBP-2 mRNA because it was the only binding protein in the anterior pituitary gland that differed in response to the administration of estradiol. Membranes were hybridized to radioactive cRNA probes for ~18 h at 65°C in prehybridization buffer containing the radioactive probe. Blots were washed twice in double-strength SSC + 0.1% SDS for 10 min at room temperature; this was followed by two 30-min washes in 0.2-strength SSC + 0.1% SDS at 65°C. Banding intensity was quantified on autoradiographs of two different exposures of each Northern blot by an LKB Bromma Ultrascan Laser Densitometer (Pharmacia LKB). Relative optical densities of the IGFBP-2 mRNA were then normalized to relative amounts of rat cyclophilin mRNA (ADU IGFBP-2/ADU rat cyclophilin) to account for unequal loading of RNA. Integrity of RNA was confirmed by visual inspection of each agarose gel under UV light after staining with ethidium bromide. The presence of the 28S and 18S ribosomal bands with no smearing between bands indicated the RNA was intact.

Statistical Analyses

The effects of estradiol on serum concentrations of LH and IGF-I and relative amounts of IGFBPs in serum were analyzed by least-squares analysis for a split-plot design using the General Linearized Model of the Statistical Analysis System [53]. Effects of estradiol treatment on tissue concentrations of hormones, IGFBPs, and relative amounts of mRNA for gonadotropin subunits and IGFBP-2 were analyzed by one-way ANOVA [53].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Mean serum concentrations of LH were higher (p < 0.05) in C ewes than in E ewes on Days 7, 30, 60, and 75 (Fig. 1). Mean serum concentrations of LH in C ewes did not differ over time (p > 0.10). However, mean serum concentrations of LH in E ewes increased (p < 0.05) with the onset of the breeding season on Days 60 and 75 compared to Days 7 and 30. Mean anterior pituitary gland concentrations of LH (1400 ± 467 and 2266 ± 555 µg/g tissue, respectively) and FSH (13.8 ± 2.4 and 18.3 ± 2.8 ng/g tissue, respectively) were similar (p > 0.10) for E and C ewes.



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  		FIG. 1. Mean serum concentrations of LH in E and C ewes on Days 7, 30, 60, and 75. Values represent least-square means ± SEM for six E ewes and four C ewes. Means with different letters differ according to treatment and (or) time (p < 0.05).

Mean serum concentrations of IGF-I were similar (p > 0.10) for E and C ewes on Day 0 (Fig. 2). By Day 7, mean serum concentrations of IGF-I were higher (p < 0.05) in E ewes than in C ewes and remained higher on Days 30, 60, and 75 (Fig. 2). Mean concentrations of IGF-I in the anterior pituitary gland were similar (p > 0.10) for E and C ewes (37.4 ± 3.3 and 41.9 ± 2.7 ng/g tissue, respectively). Mean concentrations of IGF-I in the SME were also similar (p > 0.10) in E and C ewes (24.8 ± 3.6 and 28.7 ± 4.2 ng/g tissue, respectively).



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  		FIG. 2. Mean serum concentrations of IGF-I in E and C ewes on Days 0, 7, 30, 60, and 75. Values represent least-square means ± SEM for six E ewes and four C ewes. Means with different letters differ (p < 0.05) due to treatment with estradiol.

Relative amounts of mRNA for LHß subunit in the anterior pituitary gland were higher (p < 0.05) in C ewes than in E ewes (Fig. 3). Relative amounts of mRNA for FSHß subunit and {alpha} subunit were similar (p > 0.10) in C and E ewes (Fig. 3).



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  		FIG. 3. Relative amounts of gonadotropin subunit mRNA in anterior pituitary glands from E and C ewes on Day 80. Values represent least-square means ± SEM of relative amounts of mRNA for six E ewes and four C ewes. Relative amounts of mRNA for each gonadotropin subunit were normalized to relative amounts of cyclophilin mRNA. Data are expressed in ADU. Means with different letters differ (p < 0.05).

Six IGFBPs of different molecular masses were detected in serum. On the basis of immunoprecipitation in this and previous studies [30, 45], these proteins were identified as the 24- and 28-kDa forms of IGFBP-4, the 40- and 44-kDa forms of IGFBP-3, a 34-kDa form of IGFBP-2, and an unidentified 29-kDa IGFBP. Relative amounts (ADU) of each of these IGFBPs were similar (p > 0.10) in E and C ewes on Day 0 (Table 1). Relative amounts of the 24-kDa IGFBP-4 (Fig. 4A) and 44-kDa IGFBP-3 (Fig. 4B) were higher (p < 0.05) in E ewes than in C ewes on Days 7 through 75. Relative amounts of the 40-kDa IGFBP-3 tended (p < 0.06) to be higher in E ewes than in C ewes (Fig. 4C). Quantities of the 28-kDa form of IGFBP-4 or IGFBP-2, or the 29-kDa IGFBP were not influenced (p > 0.10) by treatment or time (data not shown).


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  		TABLE 1. Relative amounts of serum IGFBPs on Day 0 in E and C ewes.a



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  		FIG. 4. Relative amounts of 24 kDa IGFBP-4 (A), 44 kDa IGFBP-3 (B), and 40 kDa IGFBP-3 (C) in serum from E and C ewes on Days 7, 30, 60, and 75. Values represent least-square means ± SEM of relative amounts of IGFBPs in six E ewes and four C ewes. Data are expressed in ADU. Means with different letters differ according to treatment (p < 0.05). * in C denotes a trend (p < 0.06) for difference between means on Day 7.

Four IGFBPs of different sizes were detected in the anterior pituitary glands and SMEs of E and C ewes. On the basis of immunoprecipitation in this and previous studies [30, 45], these proteins were identified as a 29-kDa form of IGFBP-5, 36- and 40-kDa forms of IGFBP-3, and a 32-kDa form of IGFBP-2. Relative abundance of the IGFBP-2 tended (p < 0.09) to be higher in the anterior pituitary glands of E ewes than in C ewes (Fig. 5A). This tendency was accompanied by an approximately twofold increase (p < 0.05) in mRNA for IGFBP-2 in the pituitary glands of E ewes (1.29 ± 0.16, compared to 0.62 ± 0.16 ADU). No differences were detected in relative amounts of the 36- and 40-kDa forms of IGFBP-3 or IGFBP-5 in the pituitaries of E and C ewes (Fig. 5A). Relative amounts of IGFBP-5, the 40-kDa form of IGFBP-3, and IGFBP-2 were higher (p < 0.05) in the SMEs of E ewes than of C ewes (Fig. 5B). The 36-kDa form of IGFBP-3 did not differ (p > 0.10) among E and C ewes.



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  		FIG. 5. Relative amounts IGFBPs in the anterior pituitary gland (A) and stalk median eminence (B) in E and C ewes on Day 80. Values represent least-square means ± SEM of relative amounts of IGFBPs in six E ewes and four C ewes. Data are expressed in ADU. * in A indicates a trend (p < 0.09) for difference between means. Means with different letters (B) differ (p < 0.05).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It is well established that chronic administration of low concentrations of estradiol inhibits the secretion of LH in a manner influenced by season [31, 33, 34, 54]. The onset of the anestrous season is associated with an enhanced ability of estradiol to suppress the secretion of LH, which wanes at the beginning of the breeding season ([55], current study). In non-steroid-treated ovariectomized ewes, however, the influence of season on mean serum concentrations of LH is minimal [56, 57] or not apparent ([34, 58, 59], current study).

Similar to serum LH, relative amounts of mRNA for the LHß subunit were diminished by treatment with estradiol in this and previous studies [33, 54]. The lack of detectable differences in relative amounts of mRNA for FSHß or for {alpha} subunits among estradiol-treated ewes and controls in the current study may be due to the relatively low serum concentrations of estradiol (i.e., near 5 pg/ml) in contrast to 30 pg/ml obtained in earlier studies [54].

Zeranol, a compound with estrogenic activity, increased serum levels of IGF-I in lambs [26]. Recently, components of the IGF system were identified in the ovine hypothalamus and anterior pituitary gland [29], but the effects of estradiol on the IGF-I system within these tissues were not determined. In the present experiment, treatment of ovariectomized ewes with estradiol resulted in increased circulating concentrations of IGF-I and altered the relative amounts of IGFBPs in the pituitary gland, SME, and serum. Thus, in ovariectomized ewes, estradiol altered synthesis and secretion of LH, IGF-I, and IGFBPs. It is tempting to speculate, therefore, that effects of estradiol on LH and the IGF system in sheep are evoked through common mechanisms. Circumstantial evidence in support of such a postulate was also discussed by Roberts et al. [60]. Indeed, estradiol regulates expression of the IGF-I gene [61] in chickens and expression of IGF-I and IGF-receptor in the mouse uterus [62]. In female monkeys, the sensitivity to estradiol feedback inhibition of serum LH was regulated by serum concentrations of IGF-I [63].

The elevated serum concentrations of IGF-I in estradiol-treated ewes in the present experiment is comparable to observations made in the rat [17] and in lambs treated with zeranol [26]. Elevated serum concentrations of IGF-I and increased gene expression of IGF-I in the liver occur at puberty [17], a time coincident with increased serum concentrations of estradiol [64]. Increased expression of IGF-I in the liver coincided with a tendency for higher circulating IGF levels in lambs [65]. In the rat, the liver is considered to be the most important site of IGF-I synthesis, contributing approximately 55% of circulating IGF-I [4]. However, in growing and adult swine, IGF-I mRNA abundance in skeletal and cardiac muscle [66] and the uterus [67] exceeds that in the liver. In ewes, considerable amounts of IGF-I (ng/g tissue) were found in the liver, kidney, and muscle [68]. Therefore, it is not yet clear which tissue(s) contributed the majority of IGF-I found in the serum of ewes in response to administration of estradiol.

Concomitant with the increase in circulating levels of IGF-I was an increase in relative amounts of IGFBP-3 and the 24-kDa form of IGFBP-4 in serum. Most of the IGF-I in serum circulates as a complex consisting of IGF-I, IGFBP-3, and a non-IGF binding acid labile subunit component [2]. IGFs associated with the IGFBP complex have prolonged half-lives compared to free IGF-I [69]. Elevated circulating concentrations of IGF-I are often associated with increased relative amounts of IGFBP-3 to maintain a homeostatic ratio of IGF-I to IGFBP-3. Evidence for these phenomena is that systemic administration of IGF-I to hypophysectomized animals resulted in an increase in relative amounts of circulating IGFBP-3 [70]. Thus, estradiol appears to have direct or indirect effects on circulating levels of IGFBP-3. The function of a large amount of IGF-I bound to IGFBP-3 is unknown, although several investigators have theorized that the complex may serve as a reservoir of IGF-I. During times of stress, proteases are activated to free IGF-I from the binding protein, thereby increasing IGF bioavailability [71]. The role of IGFBP-4 appears to be mainly as an inhibitor of IGF action [2] and may act to protect cells from over-stimulation by IGFs. Therefore, as estradiol increased serum concentrations of IGF-I, relative amounts of IGFBP-4 may have increased to blunt IGF-I actions. Further definition of the interactions among IGF-I and the individual IGFBPs is needed to clarify their precise physiological roles.

Although estradiol did not increase pituitary concentrations of IGF-I, it did affect relative amounts of IGFBPs in the pituitary and SME. Relative amounts of IGFBP-2 in the pituitary glands of estradiol-treated ewes tended to be higher than in controls, and relative amounts of mRNA for IGFBP-2 were higher in estradiol-treated ewes than in controls. Increased IGFBP-2 gene expression in response to estradiol was also observed in the rat pituitary [23] and the pig uterus [72]. Michels and coworkers [23] found that the highest levels of pituitary IGFBP-2 expression occurred at proestrus in the rat, a time when serum concentrations of estradiol are highest [64]. The ability of estradiol to up-regulate expression of the IGF system is, therefore, well established.

Observations that IGFBPs can function to stimulate or inhibit the actions of IGFs in a tissue-specific manner [2] indicate that the net effect (stimulatory or inhibitory) may depend on the relative amounts of the specific IGFBPs found in a tissue. In the present study, reduced levels of circulating LH were accompanied by increased levels of IGFBPs in the pituitary and SME of estradiol-treated ovariectomized ewes. There is accumulating evidence that estradiol regulates the synthesis and secretion of LH through indirect mechanisms involving nongenomic actions (reviewed in [73]). For example, gonadotropin subunit genes lack specific binding sites for the estradiol-receptor complex [73], and neurons containing GnRH do not contain receptors for estradiol (reviewed in [74]). Thus, it is tempting to speculate that estradiol may indirectly regulate the synthesis and secretion of LH through alteration of the IGF system. Additional research will be required to establish the physiological implications of these observations.

In summary, the present study demonstrates that estradiol influences components of the IGF system within the SME and pituitary gland of the ovariectomized ewe. Increased amounts of specific IGFBPs occurred in serum as well as in the pituitary gland and SME in response to estradiol. Whether the inhibitory actions of estradiol on secretion of LH are mediated by these changes in the IGF system remains to be determined. These observations provide a physiological basis for further examination and delineation of mechanisms through which specific components of the IGF system may influence reproduction in sheep.


   ACKNOWLEDGMENTS
 
We wish to thank Dr. S. Shimasaki for the plasmid containing IGFBP-2 cDNA, Dr. R. Maurer for the plasmids containing gonadotropin cDNAs, Dr. J.J. Reeves for supplying the LH antisera, and the NIDDKD for iodination-grade and standard LH and FSH as well as IGF antisera. The technical assistance of E. Van Kirk and S. Schemm is greatly appreciated.


   FOOTNOTES
 
1 This work was supported by a grant from the USDA NRICGP to G.E.M. (#9401472). Mention of trade names is necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable. Presented in part at the 88th meeting of the American Society of Animal Science, Rapid City, SD. Back

2 Correspondence: Gary E. Moss, University of Wyoming, Department of Animal Science, P.O. Box 3684, Laramie, WY 82071. FAX: (307) 766–2355; gm{at}uwyo.edu Back

3 Current address: Department of Animal & Range Sciences, South Dakota State University, Brookings, SD 57007. Back

4 Current address: USDA-CSREES-NRI, 1400 Independence Ave SW, Stop 2241, Washington, DC 20250–2241. Back

Accepted: February 25, 1998.

Received: October 14, 1997.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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