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Research Article |
Department of Biomedical Science, Colorado State University, Fort Collins, Colorado 80523
ABSTRACT
The objective of the present study was to determine how rapidly estradiol (E2) was able to suppress the secretion of LH in ovariectomized (OVX) ewes and to evaluate the ability of conjugated forms of E2 (E2 conjugated to BSA [1,3,5(10)-estratrien-3,17ß-diol-6-one-6-carboxymethyloxime:BSA [E2-BSA] and a novel conjugate, E2 conjugated to a small peptide [E2-PEP]) to mimic the actions of E2 on secretion of LH and FSH. Animals (n = 5–6 per group) were given infusions for 4 h of 50 µg of E2 or equimolar concentrations of E2-BSA or E2-PEP. Treatments with E2, E2-BSA, and E2-PEP each induced an acute suppression of LH secretion (<20 min, P < 0.01). In contrast, E2, but not E2-BSA or E2-PEP, induced the characteristic preovulatory-like surge of LH (at 10 h after priming treatment) and decreased secretion of FSH (at 4 h after priming treatment). In conclusion, the acute inhibition of LH secretion induced by E2 in OVX ewes supports the concept of a nongenomic action as the mechanism underlying the sudden suppression in secretion of LH. In addition, the fact that conjugated forms of E2 mimicked only the acute suppression of secretion of LH, without inducing the putative genomic actions on secretion of LH or FSH (i.e., a preovulatory-like surge), suggests that the acute effect of E2 may be mediated via the plasma membrane.
estradiol, estradiol receptor, follicle-stimulating hormone, luteinizing hormone, pituitary
INTRODUCTION
In the ewe and other mammalian species, estradiol (E2) has a biphasic effect on secretion of LH, characterized by a decrease in secretion followed by a preovulatory surge [1–4]. Additionally, E2 is capable of decreasing the secretion of FSH several hours after its administration [4–8]. Traditionally, the mechanism by which E2 exerts its effects has been thought to occur exclusively through regulation of the transcription of target genes. For example, the stimulatory effect of E2 on LH secretion occurs, at least in part, via an increase in the steady-state level of mRNA for GnRH receptors and number of GnRH receptors in the adenohypophysis [9–11], whereas the decrease in secretion of FSH is accompanied by an inhibition in transcription of the gene for the FSHß subunit [12, 13]. In a wide variety of biological systems, however, E2 has been shown to regulate cellular function by nongenomic actions initiated at the plasma membrane [14]. In this regard, the rapidity (within minutes) of the response induced by E2 [15] and the ability of the commercially available E2 conjugated to BSA (E2-BSA; putatively a membrane-impermeable compound) to mimic the actions of E2 [16–18] have been used widely to discriminate between genomic and nongenomic events. To our knowledge, no attempt has been made to evaluate the capability of conjugated forms of E2 to mimic the acute actions of E2 in an animal model.
The decrease in secretion of LH that is observed within a short time after administration of E2 appears to occur too rapidly to be mediated via the classic genomic action of steroid hormones [1]. Recently, we demonstrated that preincubation of primary cultures of ovine pituitary cells with E2 for 15 min prevented the GnRH-induced release of LH [19]. This effect also was observed when cells were treated with E2-BSA and a novel E2-peptide conjugate (E2-PEP). We hypothesized that E2, E2-BSA, and E2-PEP would decrease acutely the release of LH. A second hypothesis was that E2, but not the conjugated forms of E2, would induce a genomic action on gonadotropin secretion (i.e., a preovulatory-like surge) in ovariectomized (OVX) ewes.
MATERIALS AND METHODS
Preparation of Conjugated Forms of E2
Free E2 was removed from E2-BSA by six consecutive extractions with diethyl ether [20]. We previously showed that less than 1% of the weight of the conjugate is removed as free E2 and that the amount of free E2 remaining in the E2-BSA conjugate after the sixth extraction was substantially less than the concentration needed to elicit a physiological response [19]. A novel 15-amino acid sequence (N-terminus-SGGEVVVDQPMERLY-C-terminus; PEP; Macromolecular Resources) was conjugated via the N-terminal serine to 6-keto-17ß-estradiol-6-carboxy methyl oxime (E2-6-CMO; Steraloids, Inc.) by the mixed-anhydride technique [21]. Separation of E2-PEP from free E2-6-CMO was performed by Sephadex LH20 column using methanol as solvent. The 1:1 ratio of E2 and PEP in the conjugate was corroborated by Mass Spectrum Analysis (Macromolecular Resources), and conjugation efficiency was calculated based on the amount of unconjugated E2-6-CMO after separation of reactants by chromatography as described previously [19].
Animals and Experimental Protocol
All procedures involving animals were approved by the Colorado State University Animal Care and Use Committee and complied with National Institutes of Health (NIH) guidelines. Between October 2004 and January 2005, 16 mature, Western-range ewes that had been OVX for at least 2 mo were distributed randomly to receive 50 µg of E2 (n = 6) or equimolar concentrations of E2-BSA (n = 5) or E2-PEP (n = 5). Because of the variation in the number of E2 molecules attached to the original E2-BSA powder (Steraloids, Inc.), estimation of E2-BSA dosage was based on the molecular weight of BSA. All ewes were fitted with indwelling jugular cannulas (Angiocath; Becton Dickson) in the right external jugular vein to withdraw blood samples. The left external jugular vein was fitted with Silastic tubing (catalog no. 508–004; Dow Corning Corp.). This tubing was connected to a pump for infusion of the estrogens. The infusion device consisted of an autosyringe pump (Auto Syringe, Inc.) with a 30-ml plastic syringe connected to an automatic timing device allowing the delivery of 161 µl of physiological saline (0.15 M NaCl) containing 2 nmol of E2, E2-BSA, or E2-PEP every 3 min and 45 sec. To increase blood levels of E2 and the conjugates rapidly, one-third of the total dosage was injected intravenously as a priming dose. Beginning immediately after priming, the remaining dosage of each estrogen was infused intravenously over a period of 4 h. Blood samples were collected every 15 min from 4 h before to 5 h after the priming treatments. Additional blood samples were collected at 1-h intervals from 6 to 24 h after priming. Blood was allowed to clot for 1 h at room temperature and then stored at 4°C overnight. Serum was separated by centrifugation and stored at –20°C for subsequent quantification of LH [22] and FSH [23] by RIA. The reference standards for LH and FSH were NIH-oLH-S24 and NIH-oFSH-S12, respectively. Triplicate standard curves were included in each assay, and samples were analyzed in duplicate at 100 and 50 µl of sample per tube for LH and FSH, respectively. Intra-assay coefficients of variation for LH and FSH were 5% and 7%, respectively. Interassay coefficients of variation for LH and FSH were 11% and 13%, respectively. The minimum detectable dose of LH and FSH averaged 28 pg and 1.9 ng, respectively.
The following parameters were examined: 1) basal LH, defined as the lowest hormone concentration between pulses of LH; 2) mean LH, defined as the average concentration of LH; 3) number of pulses of LH, with a pulse of LH defined as a concentration of LH equal to or greater than the average concentration of LH detected during the 4-h preinfusion period and within an individual, plus 2 standard deviations, followed by at least one descending hormone concentration; 4) interval from priming of treatments to the absence of pulses of LH; and 5) length of suppression of pulsatile secretion of LH. Basal and mean LH as well as pulses of LH were determined during the 4 h before (preinfusion period) and the 4 h after (infusion period) priming treatments. The effect of treatments on the expected preovulatory-like surge of LH was evaluated using five parameters related to the massive release of LH: 1) mean LH; 2) amplitude, defined as the highest concentration of LH; 3) area under the curve during the LH surge; 4) length of preovulatory-like LH surge; and 5) interval from the priming treatment to the beginning of the massive release of LH. A preovulatory-like surge of LH was defined as a concentration of LH of at least 60 ng, which represents the estimation from several publications [2, 3], followed by at least three ascending hormone concentrations and then concentrations decreasing to pretreatment levels. Serum concentrations of FSH during the 4 h before priming treatments were averaged and compared with the hourly mean concentration of FSH observed after priming.
Data Analysis
Data were subjected to ANOVA using the general linear model of SAS [24]. Basal LH, mean LH, and number of pulses of LH, as well as changes in FSH secretion, were subjected to repeated-measures analysis. Number of pulses of LH was subjected to arcsine transformation. Sources of variation included in the model were treatment (E2, E2-BSA, and E2-PPT), period (4 h), and the interaction of treatment by period. Ewe nested in treatment was used as the error term for treatment effect. Parameters related to intervals and parameters evaluated during the preovulatory-like surge of LH were analyzed in a completely randomized design. When differences among treatment means were detected, they were separated using the least-significant-difference test adjusted by the Tukey procedure. Data are presented as mean ± SEM throughout.
RESULTS
Changes in the secretory profile of LH by treatment with E2, E2-BSA, and E2-PEP are depicted in Figures 1, 2, and 3, respectively. Before the priming treatment, basal LH, mean LH, and number of pulses of LH were similar (P > 0.2) among ewes treated with E2, E2-BSA, or E2-PEP, and no significant (P > 0.2) interaction was observed between treatment and period in any of the variables examined. Treatment with E2 or either conjugated form of E2 decreased basal LH (P < 0.05) (Fig. 4A), mean LH (P < 0.05) (Fig. 4B), and number of pulses of LH (P < 0.01) (Fig. 4C) compared to the pretreatment period. Pulses of LH were abolished rapidly after priming with E2, E2-BSA, or E2-PEP (19 ± 5 min), and no difference was found among treatments (P > 0.2) (Fig. 5A). After the initial suppression, no pulsatile activity was detected during the remainder of the infusion period, regardless of treatment (P > 0.2) (Fig. 5B).
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Regarding the effect of treatments on the induction of a preovulatory-like surge of LH, only E2 induced a massive release of LH. The surge of LH started 590 ± 32 min after priming with E2 and had a duration of 775 ± 72 min. Although E2-BSA and E2-PEP did not induce a massive release of LH, the mean, amplitude, and area under the curve of LH during the expected preovulatory-like LH surge were estimated based on the starting time and length of the LH surge observed in E2-treated ewes. The massive release of LH induced by E2 was characterized by a mean LH of 91.1 ± 8.19 ng/ml, an amplitude of 175.8 ± 12 ng/ml, and an area under the curve of 998.34 ± 122 arbitrary units. All these parameters were higher (P < 0.001) (Fig. 6) in E2-treated ewes compared with those observed during same interval in E2-BSA- and E2-PEP-treated ewes. Although conjugated forms of E2 did not induce a preovulatory-like surge of LH, the mean LH detected during the preovulatory-like surge was higher than that detected during the preinfusion period (8.99 ± 1.28 vs. 2.45 ± 1.28 ng/ml, P < 0.01).
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Before priming with the estrogens, serum concentrations of FSH were similar (490 ± 32.28 ng/ml, P > 0.2) among ewes treated with E2, E2-BSA, or E2-PEP. Treatment with E2 decreased (P < 0.01) secretion of FSH (Fig. 7A) at 4 h (503 ± 30.35 vs. 379 ± 30.2 ng/ml) after priming with E2, and FSH remained low during the next 8 h (503 ± 30.35 vs. 327 ± 30.05 ng/ml). Neither E2-BSA (Fig. 7B) nor E2-PEP (Fig. 7C) affected the secretory pattern of FSH (P > 0.1).
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DISCUSSION
In OVX ewes, infusion of E2 acutely suppressed pulsatile secretion of LH. After the initial suppression, no pulses of LH were observed for the remainder of the infusion period. The acute suppression of the pulsatile release of LH corroborates our in vitro finding that E2 decreases pituitary sensitivity to GnRH [19] and agrees with other in vivo studies in which a pituitary loci was postulated as the site for the rapid negative action of E2 [3, 4, 25]. In earlier studies, a decrease in secretion of LH was detected within an hour after E2 treatment in OVX and OVX/hypothalamo/pituitary disconnected (HPD) ewes [1, 25]. In the present study, the frequent blood sampling, together with the priming treatments, allowed a more accurate detection of the beginning of negative feedback by E2. In our hands, pulses of LH were suppressed within 20 min after beginning the infusion of E2. Because the half-life of LH in ewes is approximately 20 min [26], we did not expect to detect a suppression of pulses earlier than this. Thus, it appears that the mechanism responsible for suppression of pulses of LH was activated almost immediately after administration of the priming dose of estrogens. The sudden suppression of LH secretion is not compatible with the time necessary for a genomic action to occur.
Both E2-BSA and E2-PEP were as effective as E2 in acutely suppressing pulsatile secretion of LH and abolishing pulses of LH during the treatment period. Although the ability of E2-BSA to mimic acute actions of E2 has been demonstrated widely in vitro [16–19], this is the first report, to our knowledge, that E2-BSA and a novel synthetic E2-PEP mimic the acute action of E2 in an in vivo model. The rationale behind the use of conjugated forms of E2 as a tool to examine membrane-initiated actions is that the carrier molecule attached to E2 prevents or delays internalization of E2. Indeed, E2-BSA, distinct from E2, did not stimulate reporter activity in tumoral and nontumoral cell lines transfected with an estradiol response element-luciferase reporter construct, even when reporter activity was evaluated 18–24 h after treatment [16, 27]. These results have been interpreted as evidence that E2-BSA does not enter the cell to bind to nuclear estrogen receptors, nor does it dissociate into free E2 and BSA components. Together, these data support the concept that E2-BSA mediates its effects via the plasma membrane.
Although the hypothalamus is a recognized target for both stimulatory and inhibitory actions of E2 [28–30], it seems unlikely that conjugated forms of E2 are able to cross the blood-brain barrier. This is supported by the fact that the frequency of pulses of GnRH in OVX ewes remained unchanged during the acute negative-feedback effect of E2 on secretion of LH [31]. Therefore, the acute suppression in secretion of LH induced by E2 seems likely to occur directly at the level of the pituitary. Recently, we demonstrated that conjugated and unconjugated forms of E2 prevented the GnRH-induced secretion of LH in cultured ovine pituitary cells [19].
The mechanism by which E2 acutely suppresses secretion of LH has yet to be elucidated. In many biological systems, E2 can influence a variety of signaling pathways, several of which appear to be involved in the release of LH induced by GnRH. Specifically, E2 has been implicated in uncoupling specific G protein-coupled receptors from their effector system [32, 33], in modulation of L-type Ca2+ currents [34–36], and in nitric oxide production [37, 38].
Estradiol, but not its conjugated forms, decreased the secretion of FSH. This decrease in FSH secretion was first noted approximately 4 h after E2 priming, and concentrations of FSH remained low for the next 8 h. In the intact ewes, the half-life of FSH has been reported to range from 33 to 116 min [26, 39, 40], and although a longer half-life for FSH has been detected in OVX ewes compared with intact ewes (90 vs. 30 min) [40], the time frame for the delayed decrease in FSH is consistent with the idea of a genomic mechanism underlying the negative feedback of E2 on secretion of FSH. If E2 inhibited FSH secretion via nongenomic mechanisms, then the decrease would be expected in 90 min or less rather than after 4 h. Similar negative-feedback effects of E2 on secretion of FSH have been reported previously [4, 8]. A direct effect of E2 on secretion of FSH at the pituitary gland has been demonstrated using OVX/HPD ewes maintained with GnRH pulses [4, 8, 10] and in cultured ovine pituitary cells [6, 7]. In both in vivo and in vitro paradigms, the decrease in secretion of FSH was accompanied by a decrease in levels of FSHß mRNA in the pituitary gland [4, 8, 10, 12, 13]. Apparently, the negative action of E2 on FSHß gene expression in sheep is mediated by inhibition of pituitary ßB (activin B) gene expression [13]. The fact that conjugated forms of E2 did not induce the delayed suppression of FSH secretion observed with E2 supports the concept that conjugated forms of E2 are not capable of mimicking the genomic actions of E2.
Estradiol, but not E2-BSA or E2-PEP, induced a preovulatory-like surge of LH with characteristics similar to those reported by others [1–4]. It has been well documented that the preovulatory surge of LH, observed after administration of E2, is the result of at least two components: an increase in the number of GnRH receptors in the pituitary gland [3, 9, 11, 41], and a massive, prolonged release of GnRH from the hypothalamus [42–45]. If, as expected, conjugated forms of E2 are not able to cross the blood-brain barrier, then they would be unable to induce a GnRH surge, which could account for the lack of preovulatory-like surge of LH. In contrast, if conjugated E2 does cross the blood-brain barrier, it may indicate that attachment to a carrier molecule like BSA or a small peptide to E2 impaired the gene expression necessary for the massive release of GnRH, reaffirming the idea that conjugated forms of E2 are not capable of mimicking the classic genomic mechanism of steroid hormone action. The ability of conjugated forms of E2 to directly stimulate synthesis and/or secretion of GnRH remains to be evaluated. Recently, it was demonstrated that some, but not all, conjugated forms of E2 modulated GnRH-1 neural activity in nasal explants of mice [27]. This may be dependent on the estrogen used in the conjugation and the site on the estrogen used to attach it to BSA [27].
It is possible that the membrane-initiated actions of E2 are not restricted only to the block of GnRH-induced secretion of LH in the pituitary gland but, via activation of a distinct signal pathway and/or different plasma membrane-binding sites, may contribute to enhancement of the recruitment of transcription factors involved in the synthesis of mRNA for GnRH receptors. These combined effects of E2 (membrane plus genomic) may be responsible for the robust surge of LH in response to the E2-induced, massive release of GnRH. If activation of membrane-initiated signaling pathways are involved in the synthesis of GnRH receptors, it would explain the discrete increase in LH secretion observed during the expected preovulatory-like surge of LH in ewes treated with E2-BSA and E2-PEP. The ability of conjugated forms of E2 to stimulate synthesis of GnRH receptors remains to be tested.
Several physiological stages are observed during the reproductive life of domestic animals in which the effects of E2 on the hypothalamic-pituitary axis change from negative to positive. These changes from negative to positive feedback of E2 occur at the transition to puberty, the transition to the breeding season, and the return of cyclic ovarian activity after parturition. It is tempting to speculate that the nature of these changes may be dependent on the site of action of E2 (i.e., membrane or nuclear) in the cell during the differing physiological states.
In conclusion, the acute inhibition of secretion of LH induced by E2 in OVX ewes is not compatible with the classic genomic mechanism by which steroid hormones modulate cellular function. An acute, nongenomic action of E2 as the mechanism underlying the sudden suppression in LH secretion is supported by the fact that conjugated forms of E2 mimicked only the acute suppression of LH secretion, without inducing the putative genomic actions of E2 on FSH secretion. The presumed impermeability of conjugated forms of E2 suggests that the plasma membrane is involved in mediating the acute effect of E2.
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FOOTNOTES
1 Supported by the National Research Initiative, competitive grant no. 2005-35203-15376, from the USDA Cooperative State Research, Education, and Extension Service, and by a grant from the Colorado State University Agricultural Experiments Station. The academic program of J.A.A.-A. was supported by the National Council for Science and Technology (CONACYT-Mexico) and the National Institute for Research in Forestry, Agriculture and Livestock (INIFAP-Mexico). 
2 Correspondence: Terry M. Nett, Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Science, 3801 W. Rampart Rd., Fort Collins, CO 80523-1683. FAX: 970 491 3557; terry.nett{at}colostate.edu
Received: 15 June 2005.
First decision: 1 July 2005.
Accepted: 3 October 2005.
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