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a Institut National de la Recherche Agronomique/Unité de Recherche Associée CNRS 1291, Station de Physiologie de la Reproduction des Mammifères Domestiques, 37380 Nouzilly, France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 lower 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, 12], or activation of specific second intracellular messengers [13]. 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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. [20]. The immunization was performed according to Caraty et al. [2]. Briefly, the conjugate was dissolved in phosphate buffer and emulsified with Freund's complete adjuvant (1/3 conjugate and 2/3 adjuvant). Rams were immunized against the GnRH-BSA conjugate (n = 8) or against the carrier BSA alone (n = 2). The antigen (1 mg GnRH per 4 mg BSA) was injected intradermally (at about 30 sites) at weekly intervals for 4 wk, and intradermal booster injections were performed over 6 mo at 2-mo intervals. Antibody titer was determined by incubating serial dilutions of serum with 30 000 cpm of 125I-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 1/50 000 (n = 2) were pooled, sterilized by 0.2-µm filtration, and stored at -20°C.
Experimental Protocol
Thirteen Ile-de-France ewes that had been ovariectomized (OVX) three days beforehand were passively immunized against GnRH by 2 injections of 50 ml ovine anti-GnRH antiserum performed i.v. 3 and 6 days after ovariectomy. Three days after the last antiserum injection, the animals were randomly assigned to 3 experimental groups: group 1 (n = 3) received pulsatile saline (1 pulse per hour); groups 2 (n = 5) and 3 (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 125I, to the antiserum was demonstrated by Caraty et al. [2]. The GnRH-A (100 ng/pulse in 2 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 72 h in Bouin's Holland fixative containing 10% HgCl2 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 -20°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-µl aliquots of plasma using the RIA described by Pelletier et al. [21]. The results were expressed as nanograms LH CY1051 (equivalent to 2.5 NIH-LH-S1). The minimum detectable concentration for LH was 0.1 ng/ml. The intra- and interassay coefficients of variation for 3 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 RP2. The minimum detectable concentration for FSH was 0.2 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 µm) were incubated with rabbit anti-oLHß and horse anti-oFSHß. 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 x40 objective and a tri CCD camera (Lhesa Electronique, Cergy Pontoise, France). The analysis was carried out using the Visilog (Noesis, Velizy, France) 5.4.1 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 (20 fields were acquired per section), in two different immunocytochemistry assays.
In Situ Hybridization
In situ hybridization was performed with riboprobes prepared from cDNAs for oLHß or oFSHß cloned in pUC18 and kindly provided by R. Counis (CNRS-URA 1449; Université Pierre et Marie Curie, Paris, France). The probe encoding for oLHß was a 443-base pair (bp) fragment. The cDNA was subcloned in pGEM3zf(-) vector (Promega, Madison, WI). Plasmid containing the probe was linearized with PstI or EcoRI. Sense and antisense riboprobes were synthesized in the presence of [35S]UTP with SP6 or T7 polymerase, respectively. The probe encoding for oFSHß was a 277-bp fragment. The cDNA was subcloned in pBluescript 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 T3 polymerase, respectively.
In situ hybridization was performed as described previously [22], with minor modifications. Briefly, pituitary cryosections of 7 µm were fixed for 15 min with 4% paraformaldehyde/picric acid in PBS [23] and processed through single-strength PBS and 0.0025% Triton X-100. After being washed with PBS, the sections were progressively dehydrated (30, 50, and 70% ethanol in 0.3 M ammonium acetate) and stored at -20°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], 250 µg/ml tRNA, 2% Denhardt's reagent, 100 mg/ml PEG 6000) was applied to each section for 2 h at 50°C. For hybridization, the probes were denatured at 80°C for 5 min, diluted in hybridization buffer, and applied to the sections (2.106 cpm/30 µl 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 MgCl2 for 5 min at room temperature. They were then incubated in Tris buffer (10 mM Tris/0.5 mM NaCl, pH 8) containing 20 µg/ml ribonuclease (RNase) A twice for 30 min each at 37°C. Sections were then successively washed in 1) Tris buffer without RNase A for 30 min at 37°C; 2) buffer containing 50% formamide, 1 mM DTT, double-strength SSC for 30 min at 50°C; 3) buffer containing 50% formamide, 1 mM DTT, single-strength SSC, and 6-strength Triton X 100 for 30 min at 37°C; and 4) buffer containing 50% formamide, 1 mM DTT, 0.1-strength SSC for 30 min at 37°C. Sections were then dehydrated in ethanol (30%, 50%, and 70% in 0.3 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 in a desiccated dark box. After 30 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 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 [24]. Differences were considered significant when p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 2 (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 3 days after passive immunization and continued to decline until Day 8 (Table 2). In contrast, injection of anti-BSA serum had no effect on LH pulsatility or on FSH plasma levels (data not shown). In groups 2 and 3, pulsatile replacement of the GnRH-A restored the LH pulses, each GnRH-A injection producing an LH pulse, but failed to increase FSH levels. However, the amplitude of the induced LH pulses was lowered after 24 h of treatment in the group receiving hourly pulses (Table 1). In group 1, no LH pulses were detected after saline injection.
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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 2 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 1 = 6.9 ± 0.5% vs. group 2 = 10.5 ± 0.8%, p < 0.004, or vs. group 3 = 9.6 ± 0.4%, 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 1 = 3.7 ± 0.4% vs. group 2 = 8.4 ± 0.2%, p < 0.001, or vs. group 3 = 5.2 ± 0.8%, p < 0.03).
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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 2 = 79.2 ± 5.1% vs. group 1 = 54.6 ± 3.8%, p < 0.001; or vs. group 3 = 56.1 ± 8.9%, p < 0.001).
In Situ Hybridization
Microphotographs of pituitary sections labeled by in situ hybridization for LHß mRNA and FSHß mRNA are shown in Figure 3. Figure 4 shows the levels of LHß and FSHß mRNAs in the groups receiving high vs. low GnRH-A pulse frequency, expressed as a percentage of those observed in control immunized animals. LHß 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 FSHß mRNA levels.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 we observed is consistent with similar studies in which the endogenous GnRH input was blocked by passive immunization [2, 25], active immunization [26], or hypothalamo-pituitary disconnection [1]. The longer half-life of ovine FSH in the peripheral circulation, compared to LH, may partly explain this delay [27]. 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 72 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 [28], and the dose used for the injections was low. However, the steadiness of the amplitude between 18 and 48 h (or even 72 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 [29]. The following reduction in the amplitude of the LH pulses, also observed by Hamernik and Nett [3], 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. [30] 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 [3].
Nevertheless, the divergent patterns of LH and FSH release after passive immunization against GnRH and after 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 [26] and a reduction in the number of gonadotrophs [31]. Moreover, expression of LHß and FSHß mRNAs were lowered after hypothalamo-pituitary disconnection within 24 h [32].
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 [33].
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 LHß mRNA levels were higher after high-frequency treatment, confirming results obtained by Leung et al. [34] and suggesting a stimulatory effect of high GnRH pulse frequency on LH synthesis. Concerning FSH, the percentage of FSH-containing cells was lower under high GnRH pulse frequency, compared to low frequency, whereas FSHß 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 [35–37].
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. [38] 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 [39]. Furthermore, different cell surface densities of the GnRH-R result in a differential expression of LHß and FSHß subunit mRNAs [10]. Moreover, different second messengers may be involved in the differential regulation of LH and FSH release. An increase in intracellular Ca2+ levels in ovine pituitary cells selectively stimulates LH release, with no effect on FSH release [13]. 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 [12] or GnRH-deficient male rats [11] indicate that the decrease in FSHß 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 ß subunits as well as follistatin mRNAs in the pituitary [42], 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 33 247427743; taragnat{at}tours.inra.fr
Accepted: December 28, 1998.
Received: September 28, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, ßA and ßB subunits in various tissues predicts diverse functions. Proc Natl Acad Sci USA 1988; 85:247–251.This article has been cited by other articles:
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  J L Crawford, D A Heath, L J Haydon, B P Thomson, and D C Eckery Gene expression and secretion of LH and FSH in relation to gene expression of GnRH receptors in the brushtail possum (Trichosurus vulpecula) demonstrates highly conserved mechanisms Reproduction, January 1, 2009; 137(1): 129 - 140. [Abstract] [Full Text] [PDF]
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 L Nicol, M-O Faure, J R McNeilly, J Fontaine, C Taragnat, and A S McNeilly Bone morphogenetic protein-4 interacts with activin and GnRH to modulate gonadotrophin secretion in L{beta}T2 gonadotrophs J. Endocrinol., March 1, 2008; 196(3): 497 - 507. [Abstract] [Full Text] [PDF]
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M-O Faure, L Nicol, S Fabre, J Fontaine, N Mohoric, A McNeilly, and C Taragnat BMP-4 inhibits follicle-stimulating hormone secretion in ewe pituitary J. Endocrinol., July 1, 2005; 186(1): 109 - 121. [Abstract] [Full Text] [PDF]
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J.L. Crawford, J.R. McNeilly, and A.S. McNeilly No Evidence for Pituitary Priming to Gonadotropin-Releasing Hormone in Relation to Luteinizing Hormone (LH) Secretion Prior to the Preovulatory LH Surge in Ewes Biol Reprod, July 1, 2004; 71(1): 224 - 235. [Abstract] [Full Text] [PDF]
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S. J. Gregory, J. Townsend, A. S. McNeilly, and D. J. Tortonese Effects of Prolactin on the Luteinizing Hormone Response to Gonadotropin- Releasing Hormone in Primary Pituitary Cell Cultures During the Ovine Annual Reproductive Cycle Biol Reprod, May 1, 2004; 70(5): 1299 - 1305. [Abstract] [Full Text] [PDF]
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C. Molter-Gérard, A. Caraty, S. Guérin, J. Fontaine, and C. Taragnat Dynamic Changes in the Gonadotrope Cell Subpopulations During an Estradiol-Induced Surge in the Ewe Biol Reprod, October 1, 2000; 63(4): 1084 - 1091. [Abstract] [Full Text]
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R. C. Eagle and D. J. Tortonese Characterization and Distribution of Gonadotrophs in the Pars Distalis and Pars Tuberalis of the Equine Pituitary Gland During the Estrous Cycle and Seasonal Anestrus Biol Reprod, September 1, 2000; 63(3): 826 - 832. [Abstract] [Full Text]
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