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BOR - Papers in Press, published online ahead of print March 17, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.027615
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BIOLOGY OF REPRODUCTION 71, 224–235 (2004)
DOI: 10.1095/biolreprod.104.027615
© 2004 by the Society for the Study of Reproduction, Inc.

Pituitary

No Evidence for Pituitary Priming to Gonadotropin-Releasing Hormone in Relation to Luteinizing Hormone (LH) Secretion Prior to the Preovulatory LH Surge in Ewes1

J.L. Crawford2, J.R. McNeilly, and A.S. McNeilly

Human Reproductive Sciences Unit, Medical Research Council, University of Edinburgh Centre for Reproductive Biology, Edinburgh EH16 4SB, United Kingdom


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The purpose of this study was to determine the occurrence of and the regulatory mechanisms involved in priming of the pituitary to GnRH before the preovulatory LH surge in sheep. Experiment 1: Forty-two ewes had progestagen devices removed after 14 days and were assigned to luteal (Lut) or follicular (Foll) groups. Fifteen days later, blood sampling was initiated either immediately or 36 h after induced luteolysis in groups Lut and Foll, respectively. After 4 h, ewes were administered either saline (n = 5) or 250 ng (n = 8) or 10 µg (n = 8) of GnRH. Five ewes per treatment group were killed 1 h later, while remaining animals were blood sampled for a further 7 h. Experiment 2: Eighteen ewes were allocated to Lut and Foll groups (described above). Blood samples were collected from 2 h before GnRH (10 µg) treatment until 7 h after. Despite up-regulated GnRH-R mRNA levels in Foll ewes, pituitary content and plasma levels of LH and LHß mRNA levels were similar between groups. Mean FSHß mRNA and plasma FSH levels were elevated in Lut ewes but declined after GnRH treatment. Inversely, plasma estradiol and inhibin-A concentrations were higher in Foll ewes and declined after GnRH treatment. Fewer LH+ve/secretogranin II–ve (SgII–ve) granules were present in gonadotropes of Foll ewes, coincident with increased basal LH levels. Fewer smaller sized granules were present after GnRH treatment. In conclusion, there was no evidence of self-priming before onset of the preovulatory LH surge. Constitutive release of LH+ve/SgII–ve granules may maintain basal LH levels while smaller sized, presumably mature granules may be preferentially released after GnRH stimulation.

follicle-stimulating hormone, gonadotropin-releasing hormone, luteinizing hormone, pituitary


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Potentiation of pituitary gland's responsiveness to GnRH before the onset of the preovulatory LH surge and ovulation in women, monkeys, sheep, and rats [1–7] is thought to be due to a self-priming mechanism within the gonadotrope cell. Although this mechanism is not fully understood, it is believed to be facilitated, at least in part, through increased numbers of GnRH receptors (GnRH-R) on gonadotropes. Regulation of mRNA expression levels and subsequent numbers of GnRH-Rs are mediated through the opposing effects of estradiol (E2) and progesterone (P4). E2 up-regulated GnRH-R gene expression and subsequent concentration [3, 8–14] and modified postreceptor mechanisms that influenced LH secretion [11]. Prolonged exposure to E2 appeared necessary for maximum pituitary responsiveness to GnRH, although sustained E2 levels were not required for maintenance of the surge once initiated [15]. In contrast, addition of P4 to ovine pituitary cultures reduced GnRH-R mRNA levels and partially suppressed the upregulation induced by E2 plus inhibin [16]. Low levels of P4 at the time of onset of the preovulatory surge in rats [17] and monkeys [18] and elevated P4 levels during the luteal phase [9, 19] followed by a rapid decrease 48 h before the surge [20] in ewes appears to be necessary for maximum positive feedback action of E2 on GnRH-R mRNA levels. Furthermore, the onset of the preovulatory LH surge induces an increase in P4 in rats [21], mice [22], rhesus monkeys [23], and women [24, 25]; however, ovine preovulatory follicles appear to be incapable of synthesising P4 and thus would suggest that P4 does not play a role in the priming of gonadotropes in sheep [26].

A further possibility is that self-priming may be delayed by a functional GnRH antagonist, the putative ovarian factor gonadotropin surge-inhibiting/attenuating factor (GnSIF/AF) [27–29] which maintains the gonadotrope in a hyporesponsive, unprimed state to stimulation by GnRH. It is suggested that each endogenous pulse of GnRH results in de novo synthesis of unidentified pituitary proteins, which subsequently neutralize the inhibitory actions of GnSIF/AF [30–32]. This antagonistic seesaw continues until GnRH pulse frequency increases to approximately three pulses per hour and the inhibition by GnSIF/AF cannot overcome the stimulation by GnRH, and consequently, the LH response to GnRH increases [31], which ultimately culminates in the preovulatory LH surge. However, the identity of this putative GnSIF/AF factor remains unknown [33] and so it's role in timing of the onset of the preovulatory LH surge is unclear.

Increased binding of GnRH to its receptor during the late follicular phase [6, 8] provokes a cascade of intracellular events that assist in the amplification of the release of LH. These events involve lengthening and change in orientation of microfilaments relative to the plasmalemma [34, 35], mobilization of secretory granules to an area of membrane juxtaposed to the plasmalemma [35, 36], decrease in secretory granule size [37], potentiation of inositol 1,4,5-triphosphate and intracellular calcium mechanisms and protein kinase C [38–40], and activation of microtubule-activated protein kinase [41]. Intracellular trafficking, storage, and secretion of granules within gonadotropes may partly be facilitated through the actions of granins (secretogranin II [SgII], chromogranin A [CgA], and chromogranin B [CgB]). A close physical association between granins and gonadotropins within granules has been identified in rats [42–45], sheep [46], and mice [47]. Additionally, we have recently identified the involvement of granins in the sorting of LH into storage granules under the apparent regulation of GnRH in mice [47].

The objective of the present study was to determine whether priming of gonadotropes could be demonstrated in ewes and the role granins may play during any such period of gonadotrope self-priming. We hypothesized that the pituitary gland would be in a hyperresponsive state during the late follicular phase and that administration of GnRH would result in increased LH secretion in these animals compared with luteal ewes. Regulatory mechanisms at play during this time in comparison with the luteal phase of the estrous cycle should highlight any involvement of granins in the intracellular sorting, storage, and secretion of gonadotropins in the ewe.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental Animals

These studies were performed at the Marshall Building, Roslin, Edinburgh, UK, and all animal procedures were approved and conducted in accordance with the Home Office Animals (Scientific Procedures) Act 1996 of the United Kingdom.

Experiment 1

Forty-two Scottish black-face ewes exhibiting normal reproductive cycles were used in this study. In November, progesterone-impregnated sponges (60 mg medroxy-progesterone acetate per sponge; Intervet Laboratories Ltd., Cambridge, UK) were inserted in all animals. After 14 days in situ, sponges were withdrawn and ewes were randomly assigned to luteal (Lut; n = 21) and follicular (Foll; n = 21) groups. A blood sample was collected by jugular venepuncture from all animals 9 days after sponge withdrawal and P4 levels were measured to ensure all animals had subsequently ovulated.

Luteal ewes: On Day 15 of the subsequent estrous cycle, ewes had catheters inserted into the jugular vein and blood samples were collected every 15 min for 4 h to determine the pulsatile frequency of LH. Ewes were then administered either 250 ng (Lut-Low; n = 8) or 10 µg (Lut-Hi; n = 8) of GnRH (Sigma Chemical Co., St Louis, MO) in 1 ml physiological saline or the vehicle alone (Lut-Con; n = 5) and blood samples were collected every 10 min for 1 h. These doses of GnRH (250 ng or 10 µg) have been shown to induce a release of LH equivalent to the amplitude of an endogenous LH pulse in the early follicular phase of the cycle [48] or similar to a preovulatory LH surge [36], respectively. Five randomly selected ewes per group were immediately killed thereafter with an overdose of sodium pentobaritone (Euthatal; Rhone-Merieux Ltd., Harlow, Essex, UK) while the remaining animals (n = 3 Lut-Low and n = 3 Lut-Hi) were blood sampled every 30 min for 7 h to monitor gonadotropin secretion.

Follicular ewes: On Day 15 of the subsequent estrous cycle, luteolysis was induced in ewes by injection (i.m.) of prostaglandin F2{alpha} (PG; 100 mg cloprostenol; Estrumate; Coopers Animal Health Ltd., Crewe, Cheshire, UK) and blood samples were collected every 8 h for 32 h. Jugular catheters were then inserted in all ewes and blood samples were collected every 10 min for 4 h. At 36 h post-PG injection, ewes were administered either 250 ng (Foll-Low; n = 8) or 10 µg (Foll-Hi; n = 8) of GnRH (Sigma Chemical Co.) in 1 ml physiological saline or the vehicle alone (Foll-Con; n = 5). Blood samples were collected every 10 min for 1 h and five randomly selected ewes per group were immediately killed, while the remaining animals (n = 3 Foll-Low and n = 3 Foll-Hi) were blood sampled every 30 min for a further 7 h.

Ewes that were retained for extensive hormone profiles were not killed at the end of the experimental period. Immediately after death in all other ewes, pituitary glands were extracted and a small section was separated from the adenohypophyseal midregion for ultrastructural analysis. The remainder of the pituitary gland was divided into two pieces for storage in liquid nitrogen for molecular biological analysis. Ovaries were examined for presence of corpora lutea (CL) and stage of follicle development, size, and presence of ovulation stigma.

Experiment 2

Several ewes that were in the late follicular phase in experiment 1 underwent preovulatory LH surges before or during the sampling period. Therefore, in the following year, an additional 18 Scottish black-face ewes were included to confirm the differences in hormonal profiles found in experiment 1 between ewes in the luteal and late follicular stage after treatment with the high dose (10 µg) of GnRH. Ewes had estrous cycles synchronized exactly as described in experiment 1, progesterone-impregnated sponges were removed in November and were randomly assigned to either luteal (n = 9) or late follicular (n = 9) groups. All ewes had jugular catheters inserted immediately before the blood-sampling period.

Luteal ewes: Fifteen days after implant removal, ewes were blood sampled every 30 min for 2 h before administration of 10 µg GnRH. Blood sampling continued every 10 min posttreatment for the next hour and then every 30 min for the following 7 h.

Follicular ewes: Luteolysis was induced (described in experiment 1) 15 days after progesterone implant withdrawal and blood sampling was initiated 36 h later. The blood-sampling and treatment (10 µg GnRH) protocol was identical to that undertaken in the luteal ewes with the exception that additional daily blood samples were collected for a further 9 days after treatment to monitor P4 concentrations.

Northern Blots

Total RNA was extracted from frozen pituitary tissue and LHß, FSHß, and {alpha}-gonadotropin subunit ({alpha}GSU) mRNA levels were quantified from 15 µg total RNA samples, while mRNA levels for GnRH-R, activin-RIIB, estrogen receptor {alpha} (ER{alpha}), SgII, CgA, and CgB were quantified from 30 µg total RNA samples by Northern hybridization analysis using an appropriate 32P-labeled cDNA probe. The preparation of all the 32P-labeled cDNA probes has previously been described [8, 46, 49, 50]. Uniformity of loading was measured by reprobing the membrane with rat 18s ribosomal RNA. The intensity of the bands were quantified using a phosphorimaging system (860; Molecular Dynamics, Kent, UK) and changes in specific mRNA levels were expressed as corrected values relative to 18s rRNA levels.

Ultrastructural Studies

Small sections of pituitary glands that were assigned for analysis by transmission electron microscopy (TEM) were further divided into 1-mm3 pieces and immediately fixed and processed using a modification of a method described in Berryman et al. [51]. Modifications included the fixative (2% [w/v] paraformaldehyde, 0.1% [v/v] glutaraldehyde [25% EM-grade; Agar Scientific Ltd.], 0.2% [w/v] picric acid, 0.5 mM calcium chloride in 0.1 M phosphate buffer; pH 7.4), embedding resin (unicryl resin; British Biocell International Ltd., Cardiff, UK), and embedding capsules (gelatine capsules, size 00; Agar Scientifiic Ltd., Essex, UK).

Ultrathin sections (80 nm) were cut with a 4-mm ultradiamond knife (45°; Leica UK Ltd., Milton Keynes, UK) using a Reichert Jung Ultracut ultramicrotome (Leica UK Ltd.) and mounted on 0.25% (w/v) formvar-coated hexagonal 200 mesh gold grids (Agar Scientific Ltd.). Gonadotropes were identified by an immunogold labeling method using a monoclonal antiserum against bLHß (518B7; JF Roser, Department of Animal Science, University of California, CA) [52] and were also labeled for SgII using a rabbit-raised antiserum against bSgII (Pel-Freez Biologicals, Rogers, AR). Mounted sections were blocked with 5% (v/v) normal goat serum in Tris histochemical buffer (THB, 0.02M Tris, 0.5M sodium chloride, and 0.1% [w/v] BSA, pH 8.2) and then washed in THB. After 2 h of incubation in LH antiserum (5 µg/ml), sections were washed in THB and incubated for a further 2 h in goat anti-mouse IgG-gold particle-conjugated secondary antibody (1:50, 5-nm-diameter gold particles for monoclonal antisera; British Biocell International). Sections were washed in THB and then ddH2O. This process was repeated for double immunogold labeling using SgII primary antibody (1:1000) and goat anti-rabbit IgG-gold particle-conjugated secondary antibody (1:50; 10-nm-diameter gold particles for polyclonal antisera; British Biocell International). After the final wash step, sections were fixed in 4% (v/v) glutaraldehyde (25% EM grade) and then triple stained in Reynolds lead citrate (10 min; 0.08 M lead nitrate, 0.12 M trisodium citrate, and 0.16 M sodium hydroxide), 2% (w/v) uranyl acetate (1 h,) and again in Reynolds lead citrate (10 min), washing thoroughly in ddH2O between incubations. Sections were viewed under a Philips CM120 Biotwin TEM.

Stereological Analysis

One block of pituitary tissue was randomly selected for five animals in each of the following experimental groups: Lut-Con, Lut-Hi, Foll-Con, and Foll-Hi. Within one section from each block, every second gonadotrope cell in every second hexagonal grid slot were selected until 20 immuno gold-labeled gonadotrope cells were identified. The total number of cytoplasmic granules was counted in each of these selected cells. Additionally, in every second selected gonadotrope cell, two areas of cytoplasm (containing 10 granules each) located on opposite sides of the nucleus were selected and the numbers of LH-positive/SgII-negative (LH+ve/ SgII–ve), LH+ve/SgII+ve, and LH–ve/SgII–ve granules were counted.

Stereological determination of the size of LHß immunolabeled granules was conducted by systematic random selection [53] of 50 cells per animal group (10 cells/animal and 5 animals/group). TEM micrographs (magnifications = 28 000x) of a cytoplasmic area containing 50–200 granules were produced for each gonadotrope and then captured on a digital camera (Nikon Coolpix 990) with an image size of 2048 x 1536 pixels. All granules (minimum of 1000 granules per cell) were measured by the NIH Image 1.62 Software (by Wayne Rasband, National Institutes of Health, USA) on a Mac G4 computer. Granule profile diameter and distribution size were calculated using the Schwartz-Saltykov diameter analysis [54], which requires the granules to be spherical in shape (i.e., axial ratio {approx} 1). The calculated axial ratio for 587 randomly selected granules from five different pituitaries was 1.03 ± 0.0061. Granule diameter measurements were divided into 10 class intervals defined as equal percentage increments of the maximum observed granule diameter (420 nm).

RIA

Plasma concentrations of LH and FSH were measured by homologous RIA as described previously [55, 56] using reagents kindly supplied by Professor A.F. Parlow (NHPP, Harbor-UCLA, CA). Within this study, the limit of detection for NIH ovine LH-S18 was 0.3 ng/ml and the intra- and interassay coefficients of variation were <10%. The sensitivity of USDA-oFSH-SIAFP-RP2 (AFP 4117A) was 0.1 ng/ml and the intra- and interassay coefficients of variation were <10%. Circulating levels of E2 were measured after extraction, using reagents from MAIA estradiol kit (Serono Diagnostics, Fleet, Hants, UK) and had a sensitivity of 0.2 pg/ml and intra- and interassay coefficients of variation were <12% [57]. P4 was measured by RIA without extraction exactly as described previously [58], with a sensitivity of 0.2 pg/ml and intra- and interassay coefficients of variation were <11%. Plasma concentrations of inhibin-A were measured by ELISA as described in detail previously [49, 59], with a 30-pg/ml sensitivity and intra- and interassay coefficients of <10%. Pituitary LH and FSH content and GnRH binding levels were measured exactly as previously described [50].

Statistical Analysis

Four ewes in experiment 1 and two ewes in experiment 2 that were in the late follicular stage of the estrous cycle had preovulatory LH surges during the blood-sampling period and were eliminated from group statistical analysis. All values reported are means ± SEM. Differences in mRNA expression levels and pituitary gland content of LH and FSH were analyzed by two-way ANOVA using a generalised linear model due to unbalanced data by Minitab Statistic Software 13.31 (Microsoft Corporation). Differences in plasma concentrations of LH, FSH, E2, inhibin-A, and P4 in experiment-1 animals were analyzed using the ante-dependence analysis where possible, as in Residual Maximum Likelihood in Genstat for Windows 6th edition, Version 6.1.0.234 (VSN International Ltd., Oxford, UK). In some instances, usually after the hormone level had peaked, the autoregressive model was used because ante-dependence could not be calculated, possibly due to strong correlations. The autoregressive model assumes the same correlation between the time points while the ante-dependence model allows the correlations to change. Both methods of analysis were completed using Genstat. The parameters of the LH and FSH surges in ewes in experiments 1 and 2, LH pulse frequency and amplitude in experiment-1 ewes, and changes in granule numbers, populations, and antigenicity were analyzed using two-way ANOVA by Minitab Statistic Software 13.31. The difference between individual plasma LH and FSH concentrations at each time point in the three representative ewes that had exhibited a preovulatory LH surge (5B55, 7Y438, and BF16) compared with the values in the remaining ewes in their experimental groups that had not undergone a surge (reference ewes) were analyzed by Student t-test. Shifts in granule diameter distribution were analyzed after square-root transformation by ANOVA in Genstat.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The comparisons between ewes in the luteal or late follicular stages of the estrous cycle are referred to as stages, and the comparisons between ewes given 250 ng or 10 µg GnRH or the vehicle alone are referred to as treatments.

Pituitary-Derived mRNA Expression Levels

Experiment 1 There were no significant differences in mean LHß, {alpha}GSU, SgII, CgA (3.2-kilobase [kb], 2.8-kb, 2-kb, 1.3-kb, or ≤1-kb transcripts), CgB, ER{alpha}, and activin-R(IIB) mRNA expression levels between stages or treatments (Fig. 1). In contrast, stage and treatment effects were observed for both mean FSHß and GnRH-R (1.5-kb transcript) mRNA levels. Overall, mean FSHß expression levels were higher (P < 0.05) in luteal compared with follicular ewes and lower (P < 0.05) in ewes that received the high-dose GnRH treatment compared with low-dose GnRH treatment or saline. Mean FSHß mRNA levels (P < 0.05) were diminished in luteal ewes after the 10-µg dose of GnRH compared with all other luteal ewes. Additionally, mean FSHß mRNA levels were higher (P < 0.05) in luteal ewes administered 250 ng GnRH compared with equivalent follicular ewes (Fig. 1b). Mean mRNA expression levels for all GnRH-R transcript sizes (5.6 kb, 3.7 kb, 2.1 kb, 1.5 kb, and ≤1 kb) were elevated in follicular (P < 0.0001), compared with luteal, ewes (data not shown). Moreover, both doses of GnRH resulted in further upregulation (P < 0.05) of the 1.5-kb transcript of GnRH-R mRNA in follicular ewes only (Fig. 1h).



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  		FIG. 1. Mean changes in mRNA expression levels of (a) LHß, (b) FSHß, (c) {alpha}-gonadotropin subunit, (d) secretogranin II, (e) 2.8-kb transcript of chromogranin A, (f) chromogranin B, (g) estrogen receptor-{alpha}, (h) 1.5-kb transcript of GnRH receptor, and (i) activin receptor IIB in groups of ewes in either the luteal (black bars) or late follicular (white bars) stage of the estrous cycle at 1 h after administration of either a low (250 ng, n = 5) or high (10 µg, n = 5) dose of GnRH or the vehicle alone (control, n = 5). Values are expressed as mean ± SEM. Statistically significant differences between treatment groups are denoted by different letters

Pituitary Content of Gonadotropins

Experiment 1 Mean LH and FSH content in pituitary glands were expressed as corrected values relative to total protein content. Mean pituitary LH and FSH content and mean ratio of LH:FSH content were similar between stages and treatments (Fig. 2). However, mean pituitary contents of LH (47.35 ± 4.33 vs. 6.69 ± 0.89 ng/µg protein) and FSH (1.02 ± 0.10 vs. 0.16 ± 0.02 ng/µg protein) were diminished (P < 0.05) in those ewes that exhibited a preovulatory LH surge during the blood-sampling period although the ratio of LH:FSH content was similar to that in ewes that had not had a preovulatory surge (Fig. 2).



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  		FIG. 2. Mean changes in content of (a) LH and (b) FSH and (c) in ratio of LH:FSH content in relation to total protein content in pituitary glands of ewes in the luteal (black bars) or late follicular (white bars) stage of the estrous cycle at 1 h after administration of either a low (250 ng, n = 5) or high (10 µg, n = 5) dose of GnRH or the vehicle alone (control, n = 5). Values are expressed as mean ± SEM. The grey dotted lines represent the range of values in those late follicular ewes (n = 2) in which an endogenous preovulatory LH surge had commenced during the blood-sampling period

Plasma Concentrations of Gonadotropins, Estradiol, Inhibin-A, and Progesterone

Experiment 1 Pulse frequency of LH was threefold higher (P < 0.0005) in follicular compared with luteal ewes; however, pulse amplitude was not different between stages (Table 1). While basal LH levels appeared elevated in late follicular ewes, this was not significant due to variability of data. Mean plasma LH concentrations increased (P < 0.001) after administration of GnRH in a dose-dependent manner. The lack of difference between stages in regard to plasma LH concentrations after GnRH administration may have been due to a lack in animal numbers (n = 2–3; Fig. 3a); hence, the addition of experiment 2. Similarly, the response to GnRH in relation to both duration of LH surge and total amount of LH released were unchanged regardless of stages (Table 1). Mean plasma FSH concentrations were higher (P < 0.001) during the pre- and initial posttreatment (10–60 min) periods in luteal compared with follicular ewes, but thereafter, there was no difference. Administration of GnRH resulted in an increase (P < 0.001) in plasma FSH concentrations in a dose-dependent manner. Although the amplitude of the FSH surge was similar between stages after administration of 10 µg of GnRH, more FSH (P < 0.05) was released between 0 and 270 min post-GnRH in follicular, compared with luteal, ewes (Fig. 3b). Mean plasma E2 concentrations were higher (P < 0.001) in follicular compared with luteal ewes during the pre- and initial posttreatment (10–60 min) period, although administration of GnRH to follicular ewes resulted in a decline to levels similar to that observed in luteal ewes (Fig. 3c). Similarly, mean inhibin-A concentrations were elevated (P < 0.005) in follicular ewes during the pre- and initial posttreatment (10–60 min) period; however, this difference was negligible following administration of GnRH (Fig. 3d). Plasma P4 concentrations were elevated (2.38 ± 0.21 ng/ ml) at 9 days after sponge withdrawal in all ewes. Mean P4 concentrations during the intensive blood-sampling period (–240 to 60 min posttreatment) ranged from 2.51 ± 0.22 to 3.26 ± 0.41 ng/ml and 0.23 ± 0.12 to 0.32 ± 0.11 ng/ml in luteal and late follicular ewes, respectively.


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  		TABLE 1. Parameters of plasma LH secretion before and after treatment of GnRH (250 ng or 10 µg), in luteal and follicular ewes from Experiments 1 and 2.a



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  		FIG. 3. Mean changes in plasma levels of (a) LH, (b) FSH, (c) estradiol, and (d) inhibin A in groups of experiment-1 ewes in the luteal (black circles) or late follicular (white circles) stage of the estrous cycle that were administered either a low (250 ng, n = 8) or high (10 µg, n = 8) dose of GnRH or the vehicle alone (control, n = 5) in relation to time of administration. Values are expressed as mean ± SEM. Statistically significant differences between estrous groups are denoted by an asterisk

Experiment 2 Mean pretreatment basal LH concentrations were higher (P < 0.05) in follicular, compared with luteal, ewes (Table 1); however, posttreatment basal levels were similar (data not shown). Administration of 10 µg GnRH resulted in an immediate increase (P < 0.001) in LH concentrations although mean LH concentrations during the surge were not different between stages (Fig. 4a). Similarly, the attributes of the LH surge (mean total amount of LH released during the surge, duration, amplitude, and duration to maximum concentration) were comparable between stages (Table 1). Mean pre- and posttreatment (10– 60 min) FSH concentrations were higher (P < 0.001) in luteal compared with follicular ewes, although levels were similar after 60 min posttreatment. The total amount of FSH released during the surge was similar between stages (Fig. 4b). Mean P4 concentrations during the intensive blood-sampling period (–120 to 420 min posttreatments) ranged from 7.25 ± 1.60 to 7.82 ± 0.72 ng/ml and 0.10 ± 0.01 to 0.31 ± 0.08 ng/ml in luteal and late follicular ewes, respectively. Mean P4 concentrations progressively increased from Day 5 (0.87 ± 0.09 ng/ml) after the intensive blood-sampling period until the end of the experiment (Day 9; 4.46 ± 0.52 ng/ml) in late follicular ewes.



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  		FIG. 4. Mean changes in plasma levels of (a) LH and (b) FSH in experiment-2 ewes in the luteal (black circles, n = 9) or late follicular (white circles, n = 6) stage of the estrous cycle in relation to time of administration of 10 µg GnRH. Values are expressed as mean ± SEM. Any statistically significant differences between estrous groups are denoted by an asterisk

Natural preovulatory LH surge Three ewes that were in the late follicular stage and were treated with GnRH exhibited the onset of a preovulatory LH surge during the sampling period. Representative profiles of LH and FSH for these ewes were compared with the profile range from ewes in the same group (reference ewes) that had not yet had a preovulatory LH surge (Fig. 5). One experiment-1 ewe (5B55) was administered 250 ng of GnRH and exhibited the onset of a preovulatory LH surge at approximately 360 min after treatment. The surge in LH levels immediately following treatment in 5B55 was not different from concentrations measured in the reference ewes although plasma LH concentrations were elevated from 360 min posttreatment (Fig. 5ai). Plasma FSH concentrations in 5B55 were not different from that measured in the reference ewes (Fig. 5bi). Another experiment-1 ewe (7Y438) was administered 10 µg of GnRH and had elevated (P < 0.05) plasma LH levels at several time points during the pretreatment period compared with reference ewes. Plasma LH levels rose progressively in this animal from –80 min posttreatment until levels plateuxed at 10 min posttreatment and then started to decline by 390 min posttreatment (Fig. 5aii). Plasma FSH concentrations in 7Y438 were higher (P < 0.05) at Time 0 to 180 compared with the reference ewes (Fig. 5bii). Similarly, one experiment-2 ewe (BF16) that was in the late follicular stage and treated with 10 µg of GnRH exhibited a preovulatory LH surge at the time of GnRH administration, and plasma LH levels were markedly higher after 60 min posttreatment until the end of the sampling period compared with reference ewes, although FSH levels were similar. Interestingly, the amplitude of LH levels was higher at 60–150 min after GnRH treatment compared with that released during the sustained release period (from 180 min posttreatment) in this ewe. Two late follicular ewes in experiment 1 (ewe 7Y333 in group Foll-Con and ewe 8B040 in group Foll-Hi) displayed a preovulatory LH surge at the first intensive sampling time (–240 min) and treatment with 10 µg GnRH in ewe 8B040 at 0 min did not alter the natural progression of the LH surge (data not shown).



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  		FIG. 5. Representative profiles of plasma (a) LH and (b) FSH concentrations in three ewes in the late follicular stage that were administered either (i) 250 ng (experiment 1; ewe 5B55) or (ii, iii) 10 µg (experiment 1; ewe 7Y438 and experiment 2; BF16, respectively) of GnRH and that had a preovulatory LH surge during the blood-sampling period. The shaded regions of each graph represent each appropriate group's mean ± 2x standard deviation of hormone concentrations in ewes given the same treatment but that did not exhibit a preovulatory LH surge during the sampling period. *, Denotes that the value for each individual ewe is statistically different (P < 0.05) from the rest of the group at that time point. Note the different scale on the y-axis between corresponding graphs

Total Number and Antigenicity of Granules

The total number of granules in 80-nm-sectioned immuno-identified gonadotropes was similar between experimental groups regardless of stages or treatments (Fig. 6a). There were fewer (P < 0.05) LH+ve/SgII–ve granules within gonadotropes of follicular ewes, irrespective of treatment (Fig. 6bi). The number of LH+ve/SgII+ve and LH–ve/SgII–ve granules did not differ between stages or treatments (Fig. 6, bii and biii). There was an interaction (P < 0.05) for antigenicity of granules for LH (defined as the number of LH-conjugated gold particles/granule), whereby antigenicity was similar between stages in control animals but treatment with 10 µg GnRH resulted in an increase and decrease in antigenicity in luteal and follicular ewes, respectively (Fig. 6ci). Similarly, there was an interaction (P < 0.05) for granular antigenicity for SgII, wherein there was no difference between stages in control animals but administration of 10 µg GnRH caused a decrease and increase in antigenicity for SgII in luteal and follicular ewes, respectively (Fig. 6cii).



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  		FIG. 6. Mean change in total number of (a) granules and (b) populations of immunolabeled granules ([i] LH-positive/secretogranin II-negative [LH+ve/SgII–ve], [ii] LH+ve/SgII+ve, and [iii] LH–ve/SgII–ve) within gonadotropes, and (c) immuno-gold particles denoting (i) LH or (ii) SgII within individual granules, in 80-nm-thick pituitary sections from luteal- (black bars) and follicular- (white bars) staged ewes in experiment 1 administered either 10 µg GnRH (High) or the vehicle alone (Con). Values are expressed as mean ± SEM. Statistically significant differences between estrous groups are expressed by different letters, while a significant interaction is denoted by an asterisk

The distribution of numbers of granules per mm3 cytoplasm for each granule-size modal class was similar between luteal and late follicular ewes, but the distribution appeared to shift after treatment with 10 µg of GnRH; however, this was not significant due to variability of the data (Fig. 7). The Schwartz-Saltykov analysis demonstrated the existence of a class of granules of approximately 84–126 nm in diameter (modal class 0.3) in all control ewes; however, in similar ewes at 60 min after 10 µg of GnRH, this class of granule had disappeared. Equally, there appeared to be fewer of the next class of granules (modal class 0.4; approximately 127–168 nm in diameter) in all ewes at 60 min after GnRH treatment compared with control ewes, although again this was not significant due to variability of data. The modal class containing the largest number of granules in all groups regardless of stage or treatment was 0.5 (169–210 nm in diameter).



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  		FIG. 7. Comparison of mean ± SEM granule diameter distribution of gonadotropes, as determined by Schwartz-Saltykov analysis, in groups of ewes in the (a) luteal and (b) late follicular stage of the estrous cycle at 60 min after administration of either (i) saline or (ii) 10 µg of GnRH. Class intervals are 10% decrements of the maximum granule diameter observed (Dmax = 420 nm)

Ovarian Morphology

Ovaries were examined in all ewes killed at the end of the experimental procedure and at least one large follicle (>6 mm) as well as pale corpora albicians were present on the ovaries of all late follicular ewes. While ovaries from luteal ewes also contained numerous follicles, only one animal had a 6-mm follicle present and all ovaries contained large, red corpora lutea.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The lack of evidence for self-priming of the pituitary gland in late follicular ewes in this study was surprising considering all endocrinological and gross morphological data suggested these animals were very close indeed to the onset of the preovulatory LH surge that occurs before ovulation. This evidence includes elevated levels of E2 and inhibin-A, upregulation of GnRH-R mRNA levels, suppressed FSH concentrations, a threefold increase in LH pulse frequency, and the presence of at least one dominant, large, antral follicle (>6 mm) on the ovaries. In addition, 6/30 ewes in the late follicular phase exhibited a preovulatory LH surge during the blood-sampling procedure, verifying that the synchrony protocol used in this study resulted in the majority of late follicular ewes being as close as possible to the onset of the LH surge. While ovaries from luteal ewes also contained numerous follicles, only 1/21 animals had a 6-mm follicle present and all ovaries contained large, red corpora lutea. Despite this, mean plasma LH concentrations and the total amount of LH released after stimulation with GnRH were not different between ewes in the luteal or late follicular stage of the estrous cycle.

Interestingly, of the six ewes that exhibited a preovulatory LH surge during the sampling period, only one exhibited potentiation of the pituitary gland to a bolus of GnRH. The profile of LH release in BF16 suggests that the onset of the preovulatory LH surge occurred at the exact time of GnRH administration. The peak value of 144.6 ng/ml in this animal occurred at 90 min posttreatment and was markedly higher than LH plasma concentrations observed in any other ewe that underwent a preovulatory LH surge in this study. Additionally, plasma LH levels declined to within the normal range (30–50 ng/ml) of a preovulatory LH surge at approximately 180 min posttreatment, a similar duration to the exogenously induced surges. Plasma FSH concentrations in this animal were not different from other late follicular ewes that were administered the same dose of GnRH (10 µg), suggesting that mono-hormonal granules (i.e., LH+ve/FSH–ve) were released preferentially at this time. Interestingly, the onset of a preovulatory LH surge occurred at 360 min post-GnRH administration in ewe 5B55; however, plasma LH concentrations in this ewe were not different from other ewes in the same group in response to exogenous GnRH. Therefore, if potentiation of the pituitary gland does indeed occur before the preovulatory LH surge, it occurs sometime within 6 h before the surge. Furthermore, the onset of the preovulatory LH surge occurred at –60 min post-GnRH treatment in ewe 7Y438, which explains the elevated plasma LH concentrations at time 0–60 min posttreatment compared with the reference ewes. Plasma LH concentrations in this ewe were within the normal range for a preovulatory LH surge (≤60 ng/ml), which suggests that administration of exogenous GnRH had no effect once the surge had been initiated.

The lack of potentiation of the pituitary gland to GnRH at impending ovulation in ewes in this study suggests that the priming mechanism(s) involved in the generation of the preovulatory LH surge may be at the level of the hypothalamus rather than at the pituitary gland. Endogenous opioid peptides (EOPs) have been shown to act at the level of the hypothalamus to suppress LH release by modulating steroidal negative feedback on the hypothalamus, thereby inhibiting GnRH release in numerous mammalian species [60–66]. Consequently, perturbation of the EOP system in the ewe through administration of an opioid antagonist augments GnRH secretion into the hypophysial portal vasculature and results in a large-amplitude GnRH pulse, which subsequently causes a large-amplitude pulse of LH to be released from the pituitary gland [62]. Additionally, the content of GnRH in the hypothalamic region has been shown to change during the estrous cycle in rats, with peak content occurring on the evening of the second day of diestrus [67], at a time of increased receptivity, in relation to LH secretion, of the hypothalamus to opioid antagonists [68].

A lack of change in steady-state levels of LHß in ewes at different stages of the estrous cycle and divergence between plasma LH concentrations and LHß mRNA levels is characteristic of regulated secretory proteins due to their ability to be stored and thus released in response to ligand stimulation, in this case GnRH, without the necessity for de novo protein synthesis. These results confirm our previous observations in the ewe [8, 36, 46] of a lack of change in steady-state levels of LHß mRNA despite a reduction in LHß transcription rate in ewes at impending ovulation [69]. This disparity appears to be achieved through posttranscriptional regulation, including alterations in mRNA stability and/or translation efficiency. This is at least in part facilitated through changes in the length of the 3'-poly(A) tail [70] of LHß transcript in ewes at different stages of the estrous cycle with greater polyadenylation being prevalent in follicular, compared with luteal, ewes [46]. Conversely, FSHß mRNA levels reflect the same pattern as plasma FSH concentrations, which is representative of a protein that is predominantly secreted in a constitutive manner [26]. Both FSHß mRNA and subsequent plasma FSH levels were low in control ewes in the late follicular phase presumably due to the negative feedback effects of the elevated levels of E2 [71] and inhibin-A [72, 73] observed in these ewes. Removal of these negative feedback effects [50, 74, 75] together with augmented P4 levels [76–78] and possibly lower GnRH pulse frequency [79] was most likely to be responsible for the elevated FSHß mRNA levels and subsequent increased plasma FSH concentrations observed in the luteal-control ewes. Administration of either dose of GnRH in late follicular ewes resulted in a decline both in plasma E2 and inhibin-A concentrations to levels observed in luteal ewes and subsequently plasma FSH concentrations were also comparable between stages after GnRH stimulation. This decline in E2 is probably due at least in part to the GnRH-induced increase in plasma concentrations of LH as shown previously to occur at the onset of the natural preovulatory LH surge in the ewe [71].

Constant levels of {alpha}GSU mRNA, regardless of estrous cycle stage, supports the concept that {alpha}GSU is not rate limiting in the production of dimeric LH and FSH [80–82]. Similarly, mRNA levels of SgII, CgA, CgB, ER{alpha}, and ActRIIB were comparable between stages and after treatment with GnRH. The lack of change in granin mRNA expression levels regardless of GnRH input to the pituitary gland has also been observed in male mice [47, 83]; however, while exposure of GnRH to a mouse gonadotrope cell line (LßT2 cells) did not effect SgII mRNA levels, it has been shown to have a regulatory action on CgA mRNA levels [84]. Levels of all mRNA transcript sizes of GnRH-R were elevated in late follicular ewes compared with that in luteal ewes, as reported previously [9], and administration of 10 µg of GnRH resulted in an upregulation of the 1.5-kb transcript in luteal ewes. GnRH-R mRNA levels and subsequent numbers of GnRH-R are regulated through the stimulatory actions of E2 [6, 9, 12, 16], inhibin [9, 20], and high frequency pulses of GnRH [85, 86], and both E2 and inhibin-A concentrations as well as LH pulse frequency were shown to be elevated in the follicular phase ewes in the present study. These results suggest either that increased GnRH-R mRNA levels may not necessarily be translated into increased numbers of GnRH-Rs or that alterations in GnRH-R numbers may not be directly related to the amount of LH released in response to GnRH at any one time, but perhaps may facilitate changes in the frequency of LH release from gonadotropes.

Despite substantial amounts of LH and FSH being released during the first hour in response to a bolus of GnRH in both luteal and late follicular ewes in this study, pituitary content of gonadotropins at 1 h after GnRH treatment were similar in these ewes compared with controls. This emphasizes the capacity of gonadotrope cells to store copious amounts of both LH and FSH. Only during and after a complete preovulatory LH surge has the gonadotrope cell significantly depleted its stores of LH [8, 87], after which replenishment of storage granules containing LH was first observed sometime between 24 and 48 h postsurge [87]. A reduction in LH and FSH pituitary content was evident in the few ewes that were killed in the midst of an endogenous preovulatory surge in the present study.

Due to the lack of change in pituitary content of LH and FSH despite estrous stage and GnRH treatment, it is not surprising that the total number of storage granules in gonadotropes of ewes in this study also did not differ. However, the apparent shift in distribution of numbers of granule size classes in this study after stimulation with GnRH has been observed previously during similar physiological events in both sheep [36, 87] and mice [47]. The disappearance of all granules of 84–126 nm in diameter and the attenuation in numbers of granules of 127–168 nm in diameter in ewes at 1 h after GnRH administration suggests that it was these granules that underwent exocytosis and were secreted into the blood stream. The pathway through which nascent storage granules form begins with a protuberance of the trans-Golgi network to create a condensing vacuole [88, 89] that transforms into an immature granule capable of stimulus-dependent secretion [90, 91] only on detachment from the trans-Golgi network [92]. Maturation of an immature granule to a mature secretory granule involves an apparent reduction in volume and surface area [93] as well as an increase in dry-mass concentration [94] that results in an extremely stable electron-dense core [92], which may be separated from the membrane by a halo of space [95]. In support, we have reported that blocking GnRH input to the pituitary gland in male mice results in an unusually high proportion of electron-dense (presumably mature) granules in gonadotropes, which subsequently disappeared following stimulation with GnRH agonist [47]. We have also provided indirect evidence that the smallest sized granules were preferentially released from the storage pool in polarized (readily releasable) gonadotropes in sheep [36]. Therefore, the apparent disappearance of the smaller sized granules conforms to the hypothesis that newly formed storage granules are larger in size while the smaller sized storage granules are more mature and are in a readily releasable state for secretion into the blood stream upon stimulation of the gonadotrope cell by GnRH.

We have previously shown that intragranular associations between granins and gonadotropins appear to be regulated by GnRH in adult male mice [47]. A lack of GnRH input to the pituitary gland by immunoneutralization resulted in increased intragranular coaggregation of LH and SgII, and administration of Buserelin to these GnRH-deprived mice resulted in the subsequent disappearance of LH+ve/SgII+ve granules, presumably via exocytosis. In contrast, the numbers of LH+ve/SgII–ve granules remained unaffected by GnRH treatment, leading to the conclusion that LH+ve/SgII+ve granules were involved in the regulated secretory pathway and were therefore stored until extracellular stimulation by GnRH resulted in exocytosis, while LH+ve/SgII–ve granules were destined for constitutive release. The similarities between numbers of LH+ve/SgII+ve granules in ewes in this study regardless of stage or treatment is perhaps a reflection of both the enormous storage capacity of gonadotropes and the release of only a minute fraction of the total amount of gonadotropins stored in response to GnRH in these ewes as reflected by the lack of effect on pituitary content of LH and FSH. Basal levels of LH were significantly elevated in follicular, compared with luteal, ewes in experiment 2, and while basal LH levels appeared to be higher in follicular ewes in experiment 1, this difference was not significant due to variability of the data. These observations suggest that basal LH levels do indeed increase during the late follicular phase, and it is interesting to note that this was associated with a reduction in numbers of LH+ve/SgII–ve granules, supporting our hypothesis based on studies in the mouse [47] that these LH+ve/SgII–ve granules were released in a constitutive manner to maintain basal plasma LH concentrations. LH antigenicity was observed to increase in luteal ewes after GnRH administration and decreased in late follicular ewes after GnRH administration in this study. Interestingly, the interaction observed for antigenicity of granules for SgII was the complete reverse of this. We have previously hypothesized that coaggregation of LH with SgII results in packaging of granules for regulated secretion [47], and perhaps increased antigenic properties for SgII sensitizes these granules to secondary messenger components that are activated upon extracellular stimulation by GnRH. The change in antigenic properties of granules while LHß and SgII mRNA levels remain constant in these ewes suggests alterations in translation efficiencies of these proteins.

In conclusion, we report no evidence of self-priming of the pituitary gland in late follicular ewes as close as 6 h before the onset of the preovulatory LH surge. Potentiation of the response of the pituitary gonadotropes to GnRH was only observed in one ewe, whereby onset of the preovulatory LH surge coincided with the exact timing of exogenous administration of GnRH. This lack of self-priming of the pituitary gland was reflected in similarities in numbers of intracellular storage granules and granule populations in luteal and late follicular ewes, despite treatment with GnRH. However, there appeared to be fewer smaller sized granules in ewes that were treated with GnRH, supporting the hypothesis that granules undergoing maturation reduce in size and that mature granules are preferentially released after extracellular stimulation, while the disappearance of LH+ve/SgII–ve granules together with increased basal concentrations of LH in late follicular ewes support previous observations that these granules are released via the constitutive secretory pathway.


   ACKNOWLEDGMENTS
 
Dr. R Fischer-Colbrie (Department of Pharmacology, University of Innsbruck, Austria) kindly donated clone bovine CgB and SgII cDNA in pcDV-1 cloning vector for preparation of Northern hybridization probes and Dr. AF Parlow NIDDK generously supplied some of the reagents used in the radioimmunoassay for FSH and LH. We thank Linda Nicol, Norah Anderson, Margorie Thomson, Ian Swanston, and George Johnston for excellent technical help and Lillian Morrison for statistical analyses.


   FOOTNOTES
 
1 Supported by a postdoctoral fellowship to J.L.C. from the Journal of Reproduction and Fertility Ltd./Society for Reproduction and Fertility. Back

2 Correspondence and current address: J.L. Crawford, Reproductive Technologies Group, AgResearch Ltd., Wallaceville Animal Research Centre, Ward Street, P.O. Box 40063, Upper Hutt, New Zealand. FAX: 64 4 922 1380; janet.crawford{at}agresearch.co.nz Back

Received: 22 January 2004.

First decision: 12 February 2004.

Accepted: 4 March 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Aiyer MS, Chiappa SA, Fink G. A priming effect of luteinizing hormone releasing factor on the anterior pituitary gland in the female rat. J Endocrinol 1974 62:573-588[Medline]
  2. Fink G. Neuroendocrine control of gonadotrophin secretion. Br Med Bull 1979 35:155-160[Free Full Text]
  3. Nett TM, Crowder ME, Wise ME. Role of estradiol in inducing an ovulatory-like surge of luteinizing hormone in sheep. Biol Reprod 1984 30:1208-1215[Abstract]
  4. Fink G. The GW Harris lecture: steroid control of brain and pituitary function. J Exp Physiol 1988 73:257-293[Free Full Text]
  5. Fink G. Gonadotropin secretion and its control. In: E Knobil, Neill J (eds.), The Physiology of Reproduction. New York: Raven Press; 1988:1349–1377
  6. Turzillo AM, Campion CE, Clay CM, Nett TM. Regulation of gonadotropin-releasing hormone (GnRH) receptor messenger ribonucleic acid and GnRH receptors during the early preovulatory period in the ewe. Endocrinology 1994 135:1353-1358[Abstract]
  7. Fink G. The self-priming effect of LHRH: a unique servomechanism and possible cellular model for memory. Front Neuroendocrinol 1995 16:183-190[CrossRef][Medline]
  8. Brooks J, Taylor PL, Saunders PTK, Eidne KA, Struthers WJ, McNeilly AS. Cloning and sequencing of the sheep pituitary gonadotropin-releasing hormone receptor and changes in expression of its mRNA during the estrous cycle. Mol Cell Endocrinol 1993 94:R23-R27[CrossRef][Medline]
  9. Brooks J, McNeilly AS. Regulation of gonadotrophin-releasing hormone receptor mRNA expression in the sheep. J Endocrinol 1994 143:175-182[Abstract]
  10. Brooks J, McNeilly AS. Regulation of gonadotrophin-releasing hormone receptor expression in the ewe. Anim Reprod Sci 1996 47:89-98
  11. Clarke IJ, Cummins JT, Crowder ME, Nett TM. Pituitary receptors for gonadotropin-releasing hormone in relation to changes in pituitary and plasma gonadotropins in ovariectomized hypothalamo/pituitary-disconnected ewes. II. A marked rise in receptor number during the acute feedback effects of estradiol. Biol Reprod 1988 39:349-354[Abstract]
  12. Sealfon SC, Laws SC, Wu JC, Gillo B, Miller WL. Hormonal regulation of gonadotropin-releasing hormone receptors and messenger RNA activity in ovine pituitary culture. Mol Endocrinol 1990 4:1980-1987[Abstract]
  13. Turzillo AM, Nolan TE, Nett TM. Regulation of gonadotropin-releasing hormone (GnRH) receptor gene expression in sheep: interaction of GnRH and estradiol. Endocrinology 1998 139:4890-4894[Abstract/Free Full Text]
  14. Turzillo AM, Clapper JA, Moss GE, Nett TM. Regulation of ovine GnRH receptor gene expression by progesterone and oestradiol. J Reprod Fertil 1998 113:251-256[Abstract]
  15. Evans NP, Dahl GE, Padmanabhan V, Thrun LA, Karsch FJ. Estradiol requirements for induction and maintenance of the gonadotropin-releasing hormone surge: implications for neuroendocrine processing of the estradiol signal. Endocrinology 1997 138:5408-5414[Abstract/Free Full Text]
  16. Wu JC, Sealfon SC, Miller WL. Gonadal hormones and gonadotropin-releasing hormone (GnRH) alter messenger ribonucleic acid levels for GnRH receptors in sheep. Endocrinology 1994 134:1846-1850[Abstract]
  17. Goldman BD, Kamberi IA, Siiteri PK, Porter JC. Temporal relationship of progestin secretion, LH release and ovulation in rats. Endocrinology 1969 85:1137-1143[Medline]
  18. Neill JD, Johansson ED, Datta JK, Knobil E. Relationship between the plasma levels of luteinizing hormone and progesterone during the normal menstrual cycle. J Clin Endocrinol 1967 27:1167-1173[Medline]
  19. Caraty A, Skinner DC. Progesterone priming is essential for the full expression of the positive feedback effect of estradiol in inducing the preovulatory gonadotropin-releasing hormone surge in the ewe. Endocrinology 1999 140:165-170[Abstract/Free Full Text]
  20. Laws SC, Beggs MJ, Webster JC, Miller WL. Inhibin increases and progesterone decreases receptors for gonadotropin-releasing hormone in ovine pituitary culture. Endocrinology 1990 127:373-380[Abstract]
  21. Turgeon JL, van Patten SM, Shyamala G, Waring DW. Steroid regulation of progesterone receptor expression in cultured rat gonadotropes. Endocrinology 1999 140:2318-2325[Abstract/Free Full Text]
  22. Turgeon JL, Waring DW. Luteinizing hormone secretion from wild-type and progesterone receptor knockout mouse anterior pituitary cells. Endocrinology 2001 142:3108-3115[Abstract/Free Full Text]
  23. Remohi J, Balmaceda JP, Rojas FJ, Asch RH. The role of preovulatory progesterone in the midcycle gonadotropin surge, ovulation and subsequent luteal phase: studies with RU 486 in rhesus monkeys. Hum Reprod 1988 3:431-435[Abstract/Free Full Text]
  24. Baird DT, Thong KJ, Hall C, Cameron ST. Failure of oestrogen induced luteinizing hormone surge in women treated with mifepristone (RU486) every day for 30 days. Hum Reprod 1995 10:2270-2276[Abstract/Free Full Text]
  25. Baird DT. Antigestogens: the holy grail of contraception. Reprod Fertil Dev 2001 13:723-728[CrossRef][Medline]
  26. McNeilly AS, Crawford JL, Taragnat C, Nicol L, McNeilly JR. The differential secretion of FSH and LH: regulation through genes, feedback and packaging. Reprod Suppl 2003 61:463-476[Medline]
  27. de Koning J, Lambalk CB, Helmerhorst FM, Helder MN. Is GnRH self-priming an obligatory feature of the reproductive cycle?. Hum Reprod 2001 16:209-214[Abstract/Free Full Text]
  28. Fowler PA, Cunningham P, Fraser M, MacGregor F, Byrne B, Pappas A, Messinis IE, Templeton A. Circulating gonadotrophin surge-attenuating factor from superovulated women suppresses in vitro gonadotrophin-releasing hormone self-priming. J Endocrinol 1994 143:45-54[Abstract]
  29. Fowler PA, Templeton A. The nature and function of putative gonadotropin surge-attenuating/inhibiting factor (GnSAF/IF). Endocr Rev 1996 17:103-120[CrossRef][Medline]
  30. de Koning J. Gonadotrophin surge-inhibiting/attenuating factor governs luteinizing hormone secretion during the ovarian cycle: physiology and pathology. Hum Reprod 1995 10:2854-2861[Abstract/Free Full Text]
  31. van Dieten JAMJ, de Koning J. From basal luteinizing hormone (LH) concentrations to the preovulatory LH surge: titration of the physiological effect of gonadotrophin surge-inhibiting/attenuating factor. Hum Reprod 1995 10:3110-3116[Abstract/Free Full Text]
  32. van Dieten JAMJ, Helder MN, van den Oever C, de Koning J. Non-steroidal factors in bovine follicular fluid inhibit or facilitate the action of pulsatile administration of GnRH on LH release in the female rat. J Endocrinol 1999 161:237-243[Abstract]
  33. Fowler PA, Sorsa-Leslie T, Cash P, Dunbar B, Melvin W, Wilson Y, Mason HD, Harris W. A 60–66 kDa protein with gonadotrophin surge attenuating factor bioactivity is produced by human ovarian granulosa cells. Mol Hum Reprod 2002 8:823-832[Abstract/Free Full Text]
  34. Pickering AJ-M, Fink G. Priming effect of luteinizing hormone releasing factor in vitro: role of protein synthesis, contractile elements, Ca2+ and cyclic AMP. J Endocrinol 1979 81:223-234[Abstract]
  35. Lewis CE, Morris JF, Fink G. The role of microfilaments in the priming effect of LH-releasing hormone: an ultrastructural study using cytochalasin B. J Endocrinol 1985 106:211-218[Abstract]
  36. Currie RJW, McNeilly AS. Mobilization of LH secretory granules in gonadotrophs in relation to gene expression, synthesis and secretion of LH during the preovulatory phase of the sheep oestrous cycle. J Endocrinol 1995 147:259-270[Abstract]
  37. Lewis CE, Morris JF, Fink G, Johnson M. Changes in the granule population of gonadotrophs of hypogonadal (hpg) and normal female mice associated with the priming effect of LH-releasing hormone in vitro. J Endocrinol 1986 109:35-44[Abstract]
  38. Johnson MS, Mitchell R, Thomson FJ. The priming effect of luteinizing hormone-releasing hormone (LHRH) but not LHRH-induced gonadotropin release, can be prevented by certain protein kinase C inhibitors. Mol Cell Endocrinol 1992 85:183-193[CrossRef][Medline]
  39. Ison AJ, MacEwan DJ, Johnson MS, Clegg RA, Connor K, Mitchell R. Evidence for a distinct H7-resistant form of protein kinase C in rat anterior pituitary gland. FEBS Lett 1993 329:199-204[CrossRef][Medline]
  40. Simpson J, Johnson MS, Mitchell R. H7-resistant protein kinase C substrates in two-dimensional gels of proestrous rat anterior pituitary gland. Biochim Biophys Acta 1993 1220:69-75[Medline]
  41. Mitchell R, Sim PJ, Leslie T, Johnson MS, Thomson FJ. Activation of MAP kinase associated with the priming effect of LHRH. J Endocrinol 1994 140:R15-R18[Abstract]
  42. Watanabe T, Uchiyama Y, Grube D. Topology of chromogranin A and secretogranin II in the rat anterior pituitary: potential marker proteins for distinct secretory pathways in gonadotrophs. Histochemistry 1991 96:285-293[CrossRef][Medline]
  43. Watanabe T, Jeziorowski T, Wuttke W, Grube D. Secretory granules and granins in hyperstimulated male rat gonadotropes. J Histochem Cytochem 1993 41:1801-1812[Abstract]
  44. Watanabe T, Banno T, Jeziorowski T, Ohsawa Y, Waguri S, Grube D, Uchiyama Y. Effects of sex steroids on secretory granule formation in gonadotropes of castrated male rats with respect to granin expression. Endocrinology 1998 139:2765-2773[Abstract/Free Full Text]
  45. Watanabe Y, Azuma T, Banno T, Jeziorowski T, Ohsawa Y, Waguri S, Grube D, Uchiyama Y. Immunocytochemical localization of chromogranin A and secretogranin II in female rat gonadotropes. Arch Histol Cytol 1998 61:99-113[Medline]
  46. Crawford JL, McNeilly AS. Co-localisation of gonadotrophins and granins in gonadotropes at different stages of the oestrous cycle in sheep. J Endocrinol 2002 174:179-194[Abstract]
  47. Crawford JL, McNeilly JR, Nicol L, McNeilly AS. Promotion of intragranular co-aggregation with LH by enhancement of secretogranin II storage resulted in increased intracellular granule storage in gonadotrophs of GnRH-deprived male mice. Reproduction 2002 124:267-277[Abstract]
  48. McLeod BJ, McNeilly AS. Manipulation of plasma FSH concentrations by administration of bFF affects LH secretion in seasonally anoestrous ewes. Anim Reprod Sci 1991 25:115-124[CrossRef]
  49. McNeilly AS, Souza CJH, Baird DT, Swanton IA, McVerry J, Crawford JL, Cranfield M, Lincoln GA. Production of inhibin A not B in rams: changes in plasma inhibin A during testis growth, and expression in inhibin/activin subunit mRNA and protein in adult testis. Reproduction 2002 123:827-835[Abstract]
  50. Brooks J, Crow WJ, McNeilly JR, McNeilly AS. Relationship between gonadotrophin subunit gene expression, gonadotrophin-releasing hormone receptor content and pituitary and plasma gonadotrophin concentrations during the rebound release of FSH after the treatment of ewes with bovine follicular fluid during the luteal phase of the cycle. J Mol Endocrinol 1992 8:109-118[Abstract]
  51. Berryman MA, Rodewald RD. An enhanced method for postembedding immunocytochemical staining which preserves cell membranes. J Histochem Cytochem 1990 38:159-170[Abstract]
  52. Matteri RL, Roser JF, Baldwin DM, Lipovetsky V, Papkoff H. Characterization of a monoclonal antibody which detects luteinizing hormone from diverse mammalian species. Domest Anim Endocrinol 1987 4:157-165[CrossRef][Medline]
  53. Lucocq J. Unbiased 3-D quantitation of ultrastructure in cell biology. Trends Cell Biol 1993 3:354-358[CrossRef][Medline]
  54. Underwood EE. Quantitative Stereology. Reading, MA: Addison-Wesley Publishing Company; 1970:109–147
  55. McNeilly JR, McNeilly AS, Walton JS, Cunningham FJ. Development and application of a heterologous radioimmunoassay for ovine follicle stimulating hormone. J Endocrinol 1976 70:69-79[Abstract]
  56. McNeilly AS, Jonassen JA, Fraser HM. Suppression of follicular development after chronic LHRH immunoneutralization in the ewe. J Reprod Fertil 1986 76:481-490[Abstract]
  57. Mann GE, Lamming GE, Fray MD. Plasma oestradiol and progesterone during early pregnancy in the cow and the effects of treatment with buserelin. Anim Reprod Sci 1995 37:121-131[CrossRef]
  58. McNeilly AS, Crow WJ, Fraser HM. Suppression of pulsatile luteinizing hormone secretion by gonadotrophin-releasing hormone antagonist does not affect episodic progesterone secretion or corpus luteum function in ewes. J Reprod Fertil 1992 96:865-874[Abstract]
  59. Knight PG, Feist SA, Tannetta DS, Bleach ECL, Fowler PA, O'Brien M, Groome NP. Measurement of inhibin-A ({alpha} ßA dimer) during the oestrous cycle, after manipulation of ovarian activity and during pregnancy in ewes. J Reprod Fertil 1998 113:159-166[Abstract]
  60. Ebling FJP, Lincoln GA. Endogenous opioids and the control of seasonal LH secretion in Soay rams. J Endocrinol 1985 107:341-353[Abstract]
  61. Brooks AN, Lamming GE, Lees PD, Haynes NB. Opioid modulation of LH secretion in the ewe. J Reprod Fertil 1986 76:693-708[Abstract]
  62. Horton RJE, Cummins JT, Clarke IJ. Naloxone evokes large-amplitude GnRH pulses in luteal-phase ewes. J Reprod Fertil 1987 81:277-286[Abstract]
  63. Wu TJ, Morris DL, McArthur NH, Harms PG. Luteinizing hormone-releasing hormone (LHRH) release from ovine hypothalami is increased by the opioid antagonist naloxone in vitro. J Anim Sci 1988 67:suppl 173
  64. Yang K, Haynes NB, Lamming GE, Brooks AN. Ovarian steroid hormone involvement in endogenous opioid modulation of LH secretion in mature ewes during the breeding and nonbreeding seasons. J Reprod Fertil 1988 83:129-139[Abstract]
  65. Horton RJE, Francis H, Clarke IJ. Seasonal and steroid-dependent effects on the modulation of LH secretion in the ewe by intracerebroventricularly administered ß-endorphin or naloxone. J Endocrinol 1989 122:509-517[Abstract]
  66. Wu TJ, Morris DL, McArthur NH, Harms PG. A priming effect of naloxone on in vitro LHRH release from the hypothalamus of mid-luteal ewes. Biol Reprod 1991 44:546-549[Abstract]
  67. Chowers I, McCann SM. Content of luteinizing hormone-releasing factor and luteinizing hormone during the estrous cycle and after changes in gonadal steroid titers. Endocrinology 1965 76:700-708
  68. Petraglia F, Locatelli V, Facchinetti F, Bergamaschi M, Genazzani AR, Cocchi D. Oestrous cycle-related LH responsiveness to naloxone: effect of high oestrogen levels on the activity of opioid receptors. J Endocrinol 1986 108:89-94[Abstract]
  69. Brown P, McNeilly AS. Transcriptional regulation of pituitary gonadotrophin subunit genes. Rev Reprod 1999 4:117-124[Abstract]
  70. Bouamoud N, Lerrant