Differential Regulation of the Gonadotropin Storage Pattern by Gonadotropin- Releasing Hormone Pulse Frequency in the Ewe 1

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1 BIOLOGY OF REPRODUCTION 60, (1999) Differential Regulation of the Gonadotropin Storage Pattern by Gonadotropin- Releasing Hormone Pulse Frequency in the Ewe 1 Christine Molter-Gérard, Joel Fontaine, Sylvain Guérin, and Catherine Taragnat Institut National de la Recherche Agronomique/Unité de Recherche Associée CNRS 191, Station de Physiologie de la Reproduction des Mammifères Domestiques, 780 Nouzilly, France ABSTRACT The differential control of gonadotropin secretion by GnRH pulse frequency may reflect changes in the storage of LH and FSH. To test this hypothesis, ovariectomized ewes passively immunized against GnRH received pulsatile injections of saline (group 1) or GnRH analogue: 1 pulse/6 h for group or 1 pulse/ h for group, during 48 h. Immunization against GnRH suppressed pulsatility of LH release and reduced mean FSH plasma levels (.1 0. vs ng/ml before and days after immunization, respectively). Pulsatile GnRH analogue replacement restored LH pulses but not FSH plasma levels. Low and high frequencies of GnRH analogue increased the percentage of LH-containing cells in a similar way (group % vs. group %, or vs. group %). In contrast, the rise of the percentage of FSH-containing cells was greater after administration of the analogue at low frequency than at high frequency (group % vs. group %, or vs. group %). Moreover, while GnRH pulse frequency had no differential effect on FSH mrna levels, LH mrna levels were higher under high than low frequency. These data showed that the frequency of GnRH pulses can modulate the gonadotropin storage pattern in the ewe. These changes may be a component of the differential regulation of LH and FSH secretion. INTRODUCTION GnRH, which is secreted in pulses from the hypothalamus, plays a key role in reproductive function by regulating the biosynthesis and secretion of the gonadotropins LH and FSH. In the ewe, several studies have shown that a disruption of endogenous GnRH input by hypothalamo-pituitary disconnection or passive immunization leads to a decline of LH and FSH [1, ] and a reduction of LH and FSH mrna levels in the pituitary [, 4]. Moreover, the pulsatile character of GnRH release is essential for the stimulation of the gonadotropins, since continuous administration of GnRH desensitizes the pituitary and causes a rapid decrease in LH and FSH release [5]. Despite the fact that LH and FSH are both under the control of GnRH, their patterns of secretion are different during the estrous cycle: for example, just after ovulation, FSH release increases while plasma LH concentration remains low [6]. This differential regulation of LH and FSH may involve the frequency of GnRH pulses. Indeed, this frequency varies throughout the estrous cycle, and it increases from the luteal to the preovulatory phase [7]. In the rat, previous studies suggest that synthesis and secretion of LH are stimulated when GnRH pulse frequency is high, whereas synthesis and secretion of FSH are favored by low- Accepted December 8, Received September 8, Financially supported by a grant from the Région Centre. Correspondence. FAX: ; taragnat@tours.inra.fr 14 er GnRH pulse frequency [8, 9]. In the ewe, few data focusing on the effect of GnRH pulse frequency on gonadotropin secretion and synthesis are available. The mechanisms involved in the differential regulation of LH and FSH by GnRH pulse frequency remain unclear. They can include regulation of the GnRH receptor [10], involvement of intermediate messengers such as activin/ follistatin [11, 1], or activation of specific second intracellular messengers [1]. In addition, recruitment of specific subpopulations of gonadotrophs may be implicated. Indeed, in many species, such as the rat, pig, and sheep, there is a functional heterogeneity in the hormonal content of the gonadotrophs [14 16]. Some cells contain only one gonadotropin (monohormonal LH- or FSH-containing cells), whereas others contain both (bihormonal cells). The proportions of these different cell populations change under various physiological conditions: during the estrous cycle [17, 18], after desensitization to GnRH [18], or after GnRH exposure in vitro [19]. This dynamic heterogeneity could be part of the mechanisms occurring in the differential regulation of LH and FSH by GnRH pulse frequency. The purpose of this study, therefore, was to examine whether GnRH pulse frequency may affect the proportion of monohormonal and bihormonal cells in the ewe. MATERIALS AND METHODS Production of GnRH Antibodies Synthetic GnRH (Bale Biochem, Switzerland) was made immunogenic by conjugation to the carrier protein BSA (Sigma, l Isle d Abeau Chesnes, France) using glutaraldehyde, as previously described by Caraty et al. [0]. The immunization was performed according to Caraty et al. []. Briefly, the conjugate was dissolved in phosphate buffer and emulsified with Freund s complete adjuvant (1/ conjugate and / adjuvant). Rams were immunized against the GnRH-BSA conjugate (n 8) or against the carrier BSA alone (n ). The antigen (1 mg GnRH per 4 mg BSA) was injected intradermally (at about 0 sites) at weekly intervals for 4 wk, and intradermal booster injections were performed over 6 mo at -mo intervals. Antibody titer was determined by incubating serial dilutions of serum with cpm of 15 I-GnRH. Radiolabeled GnRH bound to the antibody was isolated by ethanol precipitation. The titer was defined as the dilution of antiserum that bound 50% of the radiolabeled GnRH. The sera of the rams whose specific GnRH-antibody titer exceeded 1/ (n ) were pooled, sterilized by 0.- m filtration, and stored at 0 C. Experimental Protocol Thirteen Ile-de-France ewes that had been ovariectomized (OVX) three days beforehand were passively immunized against GnRH by injections of 50 ml ovine anti-

2 GnRH PULSE FREQUENCY AND GONADOTROPIN STORAGE PATTERN 15 GnRH antiserum performed i.v. and 6 days after ovariectomy. Three days after the last antiserum injection, the animals were randomly assigned to experimental groups: group 1 (n ) received pulsatile saline (1 pulse per hour); groups (n 5) and (n 5) received pulsatile injections of a GnRH analogue (GnRH-A; desgly10 GnRH ethylamide, Sigma) at, respectively, 1 pulse every 6 h and 1 pulse per hour. A complete absence of binding of the GnRH-A, labeled with 15 I, to the antiserum was demonstrated by Caraty et al. []. The GnRH-A (100 ng/pulse in ml physiological saline) was injected i.v. for 48 h, and the animals were killed 1 h after the last pulse. Anterior pituitaries were immediately collected and hemisected midsagittally. One half were fixed for 7 h in Bouin s Holland fixative containing 10% HgCl for immunohistochemistry; the other half were embedded in OCT compound (Tissue-Tek; Sakura, Finetek, Torrance, CA) and stored at 70 C. Blood samples were taken before and after each injection of antiserum (every 15 min for 10 h) and during the pulsatile administration of the GnRH-A (every 15 min for 6 h on the first and second day, and just before slaughtering). The samples were heparinized and centrifuged, and the plasma was separated and stored at 0 C until assayed. Throughout the experiment, indwelling jugular catheters were used to facilitate the injections and collection of blood samples. All experiments were conducted in accordance with the guide for the care and the use of agricultural animals in agricultural research and training. RIAs LH was assayed in duplicate 100- l aliquots of plasma using the RIA described by Pelletier et al. [1]. The results were expressed as nanograms LH CY1051 (equivalent to.5 NIH-LH-S1). The minimum detectable concentration for LH was 0.1 ng/ml. The intra- and interassay coefficients of variation for plasma pools averaged 8.5% and 4%, respectively. The concentrations of plasma FSH were measured using the reagents supplied by NIADDK (Bethesda, MD), and the results were expressed as nanograms of ovine (o)fsh 19-SIAFP RP. The minimum detectable concentration for FSH was 0. ng/ml. Mean intraassay and interassay coefficients of variation were less than 9.5% and 5%, respectively. The cross reaction with olh was 0.6%. Immunocytochemistry and Quantitative Microscopical Analysis Double labeling was used to determine the percentages of LH- and FSH-containing cells (mono and bihormonal) among the total pituitary cells. It was performed as described previously [18]. Briefly, pituitary sections (7 m) were incubated with rabbit anti-olh and horse anti-o- FSH. These antibodies were revealed with goat anti-rabbit immunoglobulins conjugated to fluorescein isothiocyanate and goat anti-horse immunoglobulins conjugated to lissamine rhodamine, respectively. Light microscope images from the stained sections were acquired through a 40 objective and a tri CCD camera (Lhesa Electronique, Cergy Pontoise, France). The analysis was carried out using the Visilog (Noesis, Velizy, France) Image Analyzer. The percentage of LH- and FSH-containing cells among the total pituitary cells corresponded to the ratio of stained area: total area. The proportion of monohormonal and bihormonal cells among the gonadotrophs was determined by counting the rhodamine- and fluorescein-stained cells by eye. Four pituitary sections of each animal were treated and analyzed (0 fields were acquired per section), in two different immunocytochemistry assays. In Situ Hybridization In situ hybridization was performed with riboprobes prepared from cdnas for olh or ofsh cloned in puc18 and kindly provided by R. Counis (CNRS-URA 1449; Université Pierre et Marie Curie, Paris, France). The probe encoding for olh was a 44-base pair (bp) fragment. The cdna was subcloned in pgemzf( ) vector (Promega, Madison, WI). Plasmid containing the probe was linearized with PstI orecori. Sense and antisense riboprobes were synthesized in the presence of [ 5 S]UTP with SP6 or T7 polymerase, respectively. The probe encoding for ofsh was a 77-bp fragment. The cdna was subcloned in p- Bluescript vector SK (Stratagene, La Jolla, CA). Plasmid containing the insert was linearized with EcoRI or BamHI. Sense and antisense riboprobes were synthesized with T7 and T polymerase, respectively. In situ hybridization was performed as described previously [], with minor modifications. Briefly, pituitary cryosections of 7 m were fixed for 15 min with 4% paraformaldehyde/picric acid in PBS [] and processed through single-strength PBS and 0.005% Triton X-100. After being washed with PBS, the sections were progressively dehydrated (0, 50, and 70% ethanol in 0. M ammonium acetate) and stored at 0 C in 70% ethanol solution. Before hybridization, sections were allowed to air-dry. One hundred microliters of hybridization buffer (50% formamide, 0.6 M NaCl, 10 mm Tris, 1 mm EDTA, 1% SDS, 0.01 M dithiothreitol [DTT], 50 g/ml trna, % Denhardt s reagent, 100 mg/ml PEG 6000) was applied to each section forhat50 C. For hybridization, the probes were denatured at 80 C for 5 min, diluted in hybridization buffer, and applied to the sections (.10 6 cpm/0 l per section). Slides covered with parafilm were maintained at 50 C overnight in an humidified box. After incubation, slides were washed in PBS containing 5 mm MgCl for 5 min at room temperature. They were then incubated in Tris buffer (10 mm Tris/0.5 mm NaCl, ph 8) containing 0 g/ml ribonuclease (RNase) A twice for 0 min each at 7 C. Sections were then successively washed in 1) Tris buffer without RNase A for 0 min at 7 C; ) buffer containing 50% formamide, 1 mm DTT, double-strength SSC for 0 min at 50 C; ) buffer containing 50% formamide, 1 mm DTT, single-strength SSC, and 6-strength Triton X 100 for 0 min at 7 C; and 4) buffer containing 50% formamide, 1 mm DTT, 0.1-strength SSC for 0 min at 7 C. Sections were then dehydrated in ethanol (0%, 50%, and 70% in 0. M ammonium acetate), air-dried, and exposed to a phosphor screen for 5 days. Finally, slides were coated with autoradiographic K5 emulsion (Ilford, St Priest, France) and exposed at 4 C ina desiccated dark box. After 0 days of exposure, autoradiographies were revealed, and sections were counterstained with hematoxylin. The relative amount of mrna was assessed by densitometric scanning of the screen using an ImageQuant analyser (Molecular Dynamics, Bondoufle, France). Microscopic observation of the labeled slides allowed us to validate this quantification. Statistical Analysis All data were expressed as mean SD. Statistical comparisons of the percentages of mono- and bihormonal cells

3 16 MOLTER-GÉRARD ET AL. FIG. 1. Plasma LH and FSH levels of representative ewes from each experimental group before and after GnRH antiserum injection, and during pulsatile saline (A) or GnRH-A administration at 1 pulse/6 h (B) or 1 pulse/h (C). were performed using one-way ANOVA, followed by the Neuman-Keuls test for individual comparisons. Mean FSH plasma levels were compared by two-way ANOVA followed by Student s t-test. The parameters of LH pulse secretion were determined using the MUNRO pulse analysis program [4]. Differences were considered significant when p RESULTS Gonadotropin Secretion Plasma LH and FSH concentrations of representative ewes from each experimental group are shown in Figure 1. Characteristics of plasma gonadotropin secretion for all the animals in the study are summarized in Table 1 (LH) and Table (FSH). Passive immunization against GnRH suppressed the pulsatility of LH immediately after the anti- GnRH injection and reduced the basal LH levels (Fig. 1, Table 1). Mean FSH levels decreased within days after passive immunization and continued to decline until Day 8 (Table ). In contrast, injection of anti-bsa serum had no effect on LH pulsatility or on FSH plasma levels (data not shown). In groups and, pulsatile replacement of the GnRH-A restored the LH pulses, each GnRH-A injection producing an LH pulse, but failed to increase FSH levels. TABLE 1. Characteristics of LH pulsatility. Characteristic Group Day 0 (before anti-gnrh injection) Day 6 (am) (6 days after anti-gnrh injection) Day 6 (pm) (first GnRH-A injections) Day 8 (last GnRH-A injections) Interpulse interval (min) 1 Pulse amplitude (mg/ml) 1 Basal level (ng/ml) ** 0. 0.** ** ** * ** ** ** * p 0.05 vs. Day 0, ** p 0.01 vs. Day 0.

4 GnRH PULSE FREQUENCY AND GONADOTROPIN STORAGE PATTERN 17 TABLE. Mean FSH levels (ng/ml) SD. Group Day 0 Day Day **.1 0.**.1 0.4** * p vs. Day 0, ** p 0.05 vs. Day *.0 0.** * However, the amplitude of the induced LH pulses was lowered after 4 h of treatment in the group receiving hourly pulses (Table 1). In group 1, no LH pulses were detected after saline injection. Gonadotropin Storage Pattern Analysis of dual immunostaining showed that the gonadotrophs in GnRH-immunized OVX ewes were either monohormonal LH-containing cells or bihormonal LH/ FSH-containing cells. No monohormonal FSH-containing cell was detected. Figure shows the percentage of the gonadotroph populations among the total pituitary cells in the different experimental groups. Administration of GnRH-A at low and high frequencies induced an increase in the percentage of all cells containing LH (monohormonal LH bihormonal LH/FSH cells) and the percentage of cells containing FSH (bihormonal cells). The increase in LH-containing cells was independent of the frequency of GnRH-A pulses (group % vs. group %, p 0.004, or vs. group %, p 0.001). In contrast, the rise of FSH-containing cells was more important after administration of the analogue at low frequency than at high frequency (group % vs. group %, p 0.001, or vs. group %, p 0.0). When results were expressed as percentage of cells in the gonadotroph population, the proportion of bihormonal cells was higher in the group receiving low-frequency pulses than in other groups (group % vs. group %, p 0.001; or vs. group %, p 0.001). In Situ Hybridization Microphotographs of pituitary sections labeled by in situ hybridization for LH mrna and FSH mrna are shown in Figure. Figure 4 shows the levels of LH and FSH mrnas in the groups receiving high vs. low GnRH-A pulse frequency, expressed as a percentage of those observed in control immunized animals. LH mrna amounts were higher under high-frequency GnRH-A pulse treatment, compared to low frequency. In contrast, modification of the frequency of the pulses had no differential effect on FSH mrna levels. DISCUSSION This study focused on the effects of changes in GnRH pulse frequency on the gonadotropin storage pattern in the ewe. The experimental model we used presents the advantage of selectively blocking the action of endogenous GnRH and re-establishing a controlled pulsatile pattern with injections of the GnRH-A (desgly10 GnRH ethylamide) at chosen frequencies []. As expected, injection of GnRH antiserum dampened the pulsatile pattern of LH and reduced the mean FSH plasma levels. But whereas the release of LH was rapidly blocked, the secretion of FSH declined more gradually. The delay FIG.. Percentages of LH-containing cells (monohormonal) and LH/ FSH-containing cells (bihormonal) (mean SD) among the total pituitary cells in the experimental groups. Different letters represent statistically significant differences (p 0.05 to p 0.001). we observed is consistent with similar studies in which the endogenous GnRH input was blocked by passive immunization [, 5], active immunization [6], or hypothalamopituitary disconnection [1]. The longer half-life of ovine FSH in the peripheral circulation, compared to LH, may partly explain this delay [7]. Alternatively, these divergent patterns of LH and FSH responses suggest a slow and progressive blockade of the stimulatory mechanism of FSH release. Pulsatile GnRH-A administration after immunization restored the pulsatility of LH release, each GnRH-A injection inducing an LH pulse. However, in the ewes treated with hourly pulses of GnRH-A, the amplitude of the response was higher during the first injections, compared to amplitude of responses during the next hours. More precisely, after 18 h of treatment, the amplitude decreased and then remained constant until pituitary collection, either 48 h (reported data) or 7 h (data not shown) after the beginning of the treatment. A partial desensitization of the gonadotrophs by the GnRH-A may be the reason, although this analogue has a half-life and a biopotency in sheep comparable to that of the native decapeptide [8], and the dose used for the injections was low. However, the steadiness of the amplitude between 18 and 48 h (or even 7 h) after the beginning of the treatment does not support this hypothesis, since this down-regulation would have then been completed before 48 h of treatment. Hence, the higher amplitude of LH pulses observed at the beginning of the treatment compared to that of the following hours probably reflects the immediate LH release from intracellular pools present in the pituitary [9]. The following reduction in the amplitude of the LH pulses, also observed by Hamernik and Nett [], may represent a progressive adjustment of the pituitary gland to the exogenous treatment. After at least the first 18 hours, the maintenance of the LH pulse amplitude reflects a stabilization of the synthesis/secretion rate of LH induced by the GnRH-A. In contrast to the stimulatory effect on LH release, pulsatile GnRH-A replacement for 48 h did not restore the FSH plasma levels observed before immunization. A longer treatment with GnRH-A may be required to stimulate FSH, since Clarke et al. [0] observed an increase in FSH plasma levels in hypothalamo-pituitary-disconnected (HPD) ewes treated with hourly pulses of GnRH after 1 wk of GnRH treatment. However, this finding was not confirmed by Hamernik and Nett []. Nevertheless, the divergent patterns of LH and FSH release after passive immunization against GnRH and after

5 18 MOLTER-GÉRARD ET AL. FIG.. Photomicrographs of ovine pituitary sections labeled by in situ hybridization with 5 S antisense (A and B) or sense (C and D) riboprobes for LH (A,C) or FSH (B,D). Arrows indicate stained cells. Bar 10 m. FIG. 4. Relative quantification of LH (A) and FSH (B) mrna levels by in situ hybridization. Results are expressed as percentage of control values (group 1: 100%) SD. 48 h of GnRH-A pulses observed in our study demonstrate that the secretory mechanisms for the two gonadotropins involve different pathways. Whereas LH release is under direct control of GnRH, GnRH-independent factors may provide the primary control of FSH, GnRH being only permissive. The immunohistochemical studies showed that the gonadotrophs in GnRH-immunized OVX ewes, treated or untreated with GnRH-A, are either monohormonal LH- or bihormonal LH/FSH-containing cells. No monohormonal FSH-containing cell was detected. A similar gonadotropin storage pattern has been described in cycling ewes [18]. However, the percentages of LH- and FSH-containing cells in GnRH-immunized OVX animals are lower than those observed during estrus [18]. Indeed, suppression of GnRH input on the pituitary in OVX ewes by anti-gnrh immunization or hypothalamo-pituitary disconnection is known to induce a decrease in the pituitary content of LH and FSH within at least 7 days [6] and a reduction in the number of gonadotrophs [1]. Moreover, expression of LH and FSH mrnas were lowered after hypothalamo-pituitary disconnection within 4 h []. After suppression of endogenous GnRH by immunization, pulsatile administration of GnRH-A at both frequencies increased the percentages of all LH-containing cells (mono- and bihormonal) and FSH-containing cells (bihormonal). This enhancement reflects, at least in part, the ability of the GnRH-A to induce the synthesis of LH and FSH in pituitary cells that were not detected as gonadotrophs in the absence of GnRH-A. The failure to detect these gonadotrophs in immunized ewes may be due to low levels of hormones. Alternatively, a recruitment from a cell reserve pool and/or from another cell type may occur to produce gonadotropins in the GnRH-A-replaced ewes. Indeed, this last hypothesis was supported in the rat, in which some cells with growth hormone antigens express the gonadotropin subunit mrnas at the beginning of proestrous and may participate in the LH surge []. Changes in the frequency of the pulses had differential effects on the gonadotropin storage pattern. Concerning LH, high GnRH pulse frequency, compared to low frequency, did not affect the percentage of LH-containing cells. In contrast, the LH mrna levels were higher after high-frequency treatment, confirming results obtained by Leung et al. [4] and suggesting a stimulatory effect of high GnRH pulse frequency on LH synthesis. Concerning FSH,

6 GnRH PULSE FREQUENCY AND GONADOTROPIN STORAGE PATTERN 19 the percentage of FSH-containing cells was lower under high GnRH pulse frequency, compared to low frequency, whereas FSH mrna levels remained unchanged. These results suggest an inhibitory effect of high pulse frequency on FSH synthesis, occurring at a posttranscriptional level. These differential effects of GnRH pulse frequency could explain some of the events occurring during the estrous cycle in the intact ewe. The high GnRH pulse frequency observed at the end of the follicular phase may be responsible for an important synthesis of LH, in preparation for the massive release during the preovulatory surge. In contrast, during the luteal phase, the decrease in pulse frequency may allow the FSH storage to increase, thereby constituting a reservoir of FSH able to participate in initiation of waves of terminal follicular growth. Indeed, in the ewe, the proportion of FSH-containing cells tends to be higher during the luteal phase than during the follicular phase [18]. On the basis of the present data, the frequency of GnRH pulses selectively regulates gonadotropin storage and secretion. When the frequency of the GnRH pulses decreases, the gonadotrophs are able to switch from LH-containing cells (monohormonal) to LH/FSH-containing cells (bihormonal) by synthesizing FSH. When GnRH pulse frequency increases, the drop in FSH synthesis in these cells lets them return to monohormonal LH-containing cells. Such dynamic changes in the gonadotropin storage pattern has been observed by Childs et al. [17] in the rat during the estrous cycle. These authors suggest the existence of a gonadotroph cycle in which the maturation of the cells may involve a switch from monohormonal (nonsecreting) cells to bihormonal (secreting) cells. Furthermore, they conclude that mainly bihormonal cells participate in gonadotropin release. Previous studies performed in the sheep support this hypothesis, at least during the estrous cycle [18]. The mechanisms involved in the differential regulation of gonadotrophs still remain unclear. Since monohormonal LH-containing cells exist, they could participate in a preferential stimulation of LH by secretagogues. However, even in cells containing both LH and FSH, a differential regulation is possible, since the two hormones can be stored in separate populations of secretory granules [5 7]. At the cellular level, the divergent regulation of LH and FSH by GnRH pulse frequency could be mediated by an alteration in the number of GnRH receptors (GnRH-R). Although Clarke et al. [8] detected no differences in the expression of GnRH-R in the gonadotrophs of HPD ewes treated with pulses of GnRH at low and high frequencies, other studies showed evidence of an involvement of GnRH- R expression in the differential GnRH action. For instance, a decrease in the frequency of GnRH pulses from 1 pulse/ h to 1 pulse/4 h decreased the number of GnRH-R in castrated, testosterone-implanted rats [9]. Furthermore, different cell surface densities of the GnRH-R result in a differential expression of LH and FSH subunit mrnas [10]. Moreover, different second messengers may be involved in the differential regulation of LH and FSH release. An increase in intracellular Ca levels in ovine pituitary cells selectively stimulates LH release, with no effect on FSH release [1]. However, no specific second messenger for FSH release has been identified. The divergent regulation of LH and FSH by GnRH may also be supported by the activin/follistatin system, which can act specifically on FSH. Indeed, some studies performed on rat perifused pituitary cells [1] or GnRH-deficient male rats [11] indicate that the decrease in FSH mrna levels observed when GnRH pulse frequency increases is associated with an increase in the follistatin mrna levels. Whereas activin stimulates FSH synthesis and release [40], follistatin binds activin with high affinity and neutralizes its biological activity, thereby preventing its stimulation of FSH [41]. These factors are produced in the ovary, but recent studies detected activin subunits as well as follistatin mrnas in the pituitary [4], suggesting a possible paracrine effect of these factors on the FSH-containing cells. In conclusion, we have shown that different GnRH pulse frequencies can modulate the gonadotropin storage pattern in the gonadotrophs. Low GnRH pulse frequency enhances the storage of FSH, whereas higher frequency increases the storage of LH. These dynamic changes may participate in the differential regulation of the gonadotropins. ACKNOWLEDGMENTS The authors wish to thank Dr. Y. Combarnous (INRA, Nouzilly, France), for providing purified hormones, Dr. R. Counis (CNRS, Université Pierre et Marie Curie, Paris, France) for the LH and FSH probes, the NIH Pituitary Hormone Distribution Program (Baltimore, MD) for FSH RIA reagents, Dr. Y. Tillet (INRA, Nouzilly, France) for providing anti-fsh serum, and O. Bastien (INRA, Jouy, France) for expert assistance in image analysis. They also thank A. Caraty (INRA, Nouzilly, France) and Dr. D. Monniaux (INRA, Nouzilly, France) for helpful discussion. REFERENCES 1. Clarke IJ, Cummins JT, de Kretser DM. Pituitary gland function after disconnection from direct hypothalamic influences in the sheep. Neuroendocrinology 198; 6: Caraty A, Martin GB, Montgomery G. 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