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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 6 2934-2942
Copyright © 2003 by The Endocrine Society

Developmental Changes in the Hormonal Identity of Gonadotroph Cells in the Rhesus Monkey Pituitary Gland

Dawud Meeran, Henryk F. Urbanski, Susan J. Gregory, Julie Townsend and Domingo J. Tortonese

Department of Anatomy (D.M., S.J.G., J.T., D.J.T.), University of Bristol, Bristol BS2 8EJ, United Kingdom; and Division of Neuroscience, Oregon National Primate Research Center (H.F.U.), Beaverton, Oregon 97006

Address all correspondence and requests for reprints to: Dr. Domingo J. Tortonese, Department of Anatomy, University of Bristol, Southwell Street, Bristol BS2 8EJ, United Kingdom. E-mail: d.tortonese{at}bristl.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To help elucidate the regulatory mechanism responsible for divergent gonadotrophin secretion during sexual maturation, we examined the gonadotroph population and hormonal identity of gonadotroph subtypes in pituitary glands of juvenile (age, 1.7 ± 0.2 yr) and adult (age, 12.3 ± 0.8 yr) male rhesus monkeys (Macacca mulatta). Serum LH and testosterone concentrations were, respectively, 3 and 7 times lower in juveniles than in adults, thus confirming the different stages of development. Immunohistochemistry revealed that the proportion of LH gonadotrophs in relation to the total pituitary cell population in the juvenile animals was significantly smaller than in the adults. In a subsequent study, double immunofluorescent labeling identified three distinct gonadotroph subtypes in both age groups: ones expressing either LH or FSH and another one expressing a combination of both gonadotrophins. Whereas the number of monohormonal LH cells per unit area was greater in the adults than in the juveniles, the number of monohormonal FSH gonadotrophs was remarkably lower. However, the proportion of FSH cells (whether mono- or bihormonal) within the gonadotroph population was similar between groups. Interestingly, the proportion and number of bihormonal gonadotrophs as well as the LH/FSH gonadotroph ratio were significantly greater in the adults than in the juveniles. Taken together, these data reveal that during the juvenile-adult transition period, not only does the pituitary gonadotroph population increase, but a large number of monohormonal FSH gonadotrophs are likely to become bihormonal. Because this morphological switch occurs when marked changes in plasma gonadotrophins are known to occur, it may represent an intrapituitary mechanism that differentially regulates gonadotrophin secretion during sexual development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE IDEA THAT a single hypothalamic peptide, GnRH, regulates the secretion of the two gonadotrophins, LH and FSH, from the pituitary gland is widely accepted. However, considering the numerous episodes of divergent gonadotrophin secretion observed physiologically, a mechanism capable of differentially regulating the secretion of LH from that of FSH is likely to operate within the pituitary. At puberty, differential changes in gonadotrophin secretion cause the dramatic switch from an FSH- to an LH-dominated pattern (1, 2), whereas reversion to FSH dominance occurs at the menopause (1). Species with a seasonal breeding pattern, including the rhesus monkey, exhibit a similar divergence in gonadotrophin secretion between breeding and nonbreeding seasons (3). Divergence in the pattern of gonadotrophins in photoperiodic species is also observed in a shorter time frame, i.e. during the estrous cycle; following ovulation, plasma concentrations of FSH increase, but those of LH decline and remain low (4, 5).

Differences between the fertile and infertile patterns of hypothalamic and gonadal activity are likely to contribute to the differential control of gonadotrophins. For example, the preferential stimulation of LH-ß mRNA transcription by high-frequency pulsatile GnRH (6), hormone-specific sensitivity to the effects of gonadal steroids (7), and the selective action of inhibin (8) all are likely to play a role in the differential regulation of gonadotrophin secretion. Moreover, specific changes in the anatomical relationship between gonadotrophs and other pituitary cell types may contribute to this regulation by modifying local paracrine signals from one cell type to another (9, 10). However, regardless of the relative contributions made by these various potential signaling factors, the pituitary gonadotroph population, as the site of gonadotrophin synthesis, storage, and secretion, must comprise the effector mechanism by which differential changes in secretion are achieved. Comparison of the gonadotroph populations between stages of fertility and infertility may contribute to unravel the nature of the effector mechanism.

Early studies in the rat showed changes not only in the morphological features but also in the relative proportions of pituitary cells during postnatal development (11). It is now well accepted, that the high-frequency pulsatile pattern of GnRH secretion characteristic of the fertile state stimulates gonadotroph differentiation (12) and could contribute to differential changes in gonadotrophin secretion by causing a relatively greater increase in the population of LH-secreting gonadotrophs. This is feasible because the existence of functional subsets of gonadotrophs, which respond to GnRH by secreting LH and FSH in different proportions (12), has been reported. Indeed, studies in the rat (13, 14, 15, 16), sheep (17, 18), horse (19, 20), and humans (21, 22) all demonstrated heterogeneity in the pituitary gonadotroph population and the existence of LH-monohormonal, FSH-monohormonal and bihormonal gonadotrophs subtypes. Changes in the proportion of gonadotrophs in each subset, perhaps in response to the pattern of GnRH and/or gonadal steroid input, could enable gonadotrophin secretion to be differentially controlled by a single hypothalamic hormone (15, 23, 24). Cytophysiological observations in the rat and sheep provide support for this theory. Changes in the populations of gonadotroph subtypes, which may contribute to the aforementioned divergence in the patterns of gonadotrophins, were detected during the estrous cycle (18, 25) and in response to changes in GnRH pulse frequency (26). Moreover, the heterogeneity of gonadotrophin storage in the rat pituitary showed dynamic modifications during sexual maturation (27). In the current study, we examined the primate pituitary and specifically investigated developmental changes in the gonadotroph population of the rhesus monkey pituitary gland as possible morphological basis for the differential control of gonadotrophin secretion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and tissues

Developmental changes in the hormonal identity of primate pituitary gonadotrophs were evaluated by comparing the gonadotroph populations in the pituitary glands of juvenile (age, 1.7 ± 0.2 yr; n = 5) and adult (age, 12.3 ± 0.8 yr; n = 5) male rhesus monkeys (Macaca mulatta). Animal care was provided by the Oregon National Primate Research Center (ONPRC) in accordance with the NIH Guide for the Care and Use of Laboratory Animals. The animals were painlessly killed as part of the ONPRC Tissue Distribution Program to provide tissue for this and other unrelated studies. The animals were not subjected to any prior treatment and were euthanized according to procedure recommended by the Panel on Euthanasia of the American Veterinary Association. Each animal was sedated with ketamine and then given an overdose of pentobarbital (25 mg/kg, iv). The pituitary glands were fixed in Bouin’s solution for 36 h at room temperature and then transferred into 70% ethanol. They were sectioned through either the transverse or sagittal plane before half of each pituitary was dehydrated in solutions of 70% methanol, then 100% ethanol, and finally placed in cleaning solvent (Histoclear; Raymond Lamb Ltd., Eastbourne, East Sussex, UK) before being embedded in paraffin wax using an automatic processor (Shandon 2LE, Life Sciences International, Basingstoke, Hampshire, UK).

Hormone assays

At death, blood samples were collected to measure the concentrations of LH and testosterone in the peripheral circulation. Bioactive serum concentrations of LH were determined using a previously validated mouse Leydig cell bioassay that involves RIA for testosterone (28); this procedure is routinely performed by the ONPRC Assay Core because it requires only a small volume of serum (<10 µl). Serum testosterone concentrations were determined by RIA as previously described (29). The effects of reproductive state (juvenile vs. adult) on hormone concentrations were examined by unpaired t test with Welch’s correction for unequal variance.

Experiment 1: nonfluorescent staining for determination of incidence and distribution of gonadotrophs

Nonfluorescent staining was performed using a monoclonal antibody to the bovine LH ß-subunit (518 B7; a gift from Dr. J. F. Roser, University of California-Davis). Pituitary sections (6 µm) were cut, mounted onto vectorbond-coated (Vector Laboratories Ltd., Peterborough, UK) slides, and incubated overnight at room temperature. Sections were then dewaxed, rehydrated, and washed in Tris-buffered saline (TBS; 0.05 M, pH 7.4). After inhibition of endogenous peroxidase activity by incubation for 20 min in 3% hydrogen peroxide in methanol, sections were blocked overnight at 4 C in normal rabbit serum (1:5 dilution in TBS, DAKO Corp., Ely, UK). Subsequently, LH-ß antibody (1:1350 dilution in TBS) was applied to each section for 2 h at room temperature. For negative control sections, the primary antibody was omitted. Sections were then rinsed (3 x 5 min) in 0.1 M PBS containing 0.1% BSA (PBS-BSA) and incubated with rabbit antimouse serum conjugated to horseradish peroxidase (1:200 dilution in PBS-BSA, DAKO Corp.) for 2 h at room temperature. Sections were rinsed (3 x 10 min) in PBS-BSA, and bound antibody was detected using 3,3'-diaminobenzidine (DAKO Corp.). All sections were counterstained with hematoxylin, dehydrated, coverslip mounted using DePex (Merck Ltd., Poole, UK), and visualized by light microscopy.

Quantitative and statistical analyses of data from nonfluorescent staining

Using at least two separate immunocytochemistry runs, three pituitary sections per animal were stained to identify and count gonadotroph cells. LH-immunopositive and counterstained cells were counted from five fields per section within the pituitary pars distalis (PD), totaling 15 fields counted per animal. Field dimensions were defined by a 10-mm2 graticule, used at a magnification of x200 under a light microscope (Leitz, Stuttgart, Germany). The graticule divided the field into 100 equally sized quadrants, of which 20 were randomly chosen to be counted from. Because of the regional variation in LH-gonadotroph distribution, it was necessary to count from the same set of regions in each section. Of the five fields chosen, three were taken from the rostral border of the PD, one from the center, and one from the PD region that is adjacent to the pars intermedia (PI). Cells other than LH-gonadotrophs were stained blue, whereas gonadotrophs appeared brown, allowing them to be easily identified. Photographs were taken to support quantitative data and corroborate the changes in distribution that were observed. The effects of reproductive state (i.e. juvenile vs. adult) on the number of gonadotrophs/field and proportion of gonadotrophs in relation to the total pituitary cell population were examined by ANOVA.

Experiment 2: immunofluorescent staining for determination of gonadotroph subtypes

Immunofluorescent staining for LH and FSH was carried out following a method described previously (17). Tissue sections (6 µm) were cut, mounted onto vectorbond-coated slides, and dewaxed, rehydrated and rinsed as described above. Nonspecific binding sites were blocked overnight at 4 C with normal goat serum and/or normal donkey serum (1:5 dilution in TBS, Vector Laboratories). For double immunostaining, 50 µl LH-ß monoclonal antibody (518 B7, 1:500 in TBS) were used in combination with 50 µl of a rabbit polyclonal antibody to the human FSH-ß subunit (M91, 1:200 in TBS; a gift from Prof. A. S. McNeilly, MRC Human Reproductive Sciences Unit, Edinburgh, UK). For single stainings, primary antibodies were applied at the same working dilutions. Negative controls included omission of primary antibody, and replacement of primary antibody by the appropriate serum. Sections were incubated with primary antibodies for 1 h at room temperature and then overnight at 4 C in a humidity chamber; the sections were then washed (3 x 5 min) in PBS-BSA before incubation with secondary antibodies. For double staining, sections were incubated sequentially with goat antimouse serum conjugated to rhodamine (Sigma Chemical Co., Poole, Dorset, UK) and donkey antirabbit serum conjugated to fluorescein (Diagnostics Scotland, Lanarkshire, UK), both diluted 1:20 in PBS-BSA, for 1 h (each) at room temperature, with a PBS-BSA wash in between. For single stainings, only one of the two secondary antibodies was used at the same working dilution. Finally, sections were rinsed (5 x 5 min) in PBS-BSA and mounted using fluorescent mounting medium (Vectashield, Vector Laboratories).

Qualitative, quantitative, and statistical analyses of data from fluorescent staining

Using at least two separate immunocytochemistry runs, three pituitary sections per animal were stained to identify LH-ß and FSH-ß immunopositive cells. The hormonal identity of cells was determined by their fluorescence under two separate wavelengths of UV light when viewed under the microscope (Leica, Wetzlar, Germany). Immunopositive cells were counted from two fields per pituitary section, totaling six fields counted per animal. The field area was defined by a microscope graticule rectangle, which corresponded to the photographic field. For each field (370 x 240 µm when viewed at a magnification of x400), the total number of LH- and FSH-immunopositive cells was counted directly from the microscope. To count bihormonal gonadotrophs, double-exposed photographs were taken for each field counted with a camera (Leica) and AS-400 slide film (Kodak, Rochester, NY). Because the same photographic frame had been exposed to both wavelengths, these photographs showed all gonadotrophs, the hormonal identity of which could be discriminated by their color. Whereas LH-monohormonal gonadotrophs appeared red and FSH-monohormonal cells appeared green, bihormonal cells were orange-yellow. Cell counts were checked for accuracy by repetition. As bihormonal cells fluoresced at both wavelengths, these cells were counted twice in each field. Therefore, to determine the total number of gonadotrophs in each field, the bihormonal number was deducted from the combined totals of LH- and FSH-immunopositive cells. To determine the number of LH- and FSH-monohormonal gonadotrophs in each field, the number of bihormonal cells was deducted from the individual totals of LH- and FSH-positive cells, respectively. Single- and double-exposed photographs were taken to support quantitative data and to corroborate the changes in hormonal identity that were observed. The effects of reproductive state (i.e. juvenile vs. adult) on number of cells/field and proportion of gonadotroph subtypes in relation to the total gonadotroph population, as well as on the LH/FSH gonadotroph ratio, were examined by ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Preliminary staining and gonadotroph distribution in the primate pituitary gland

Pilot studies determined the optimal final working concentrations of the antibodies used: LH-ß at 1:1350 for nonfluorescent staining and at 1:1000 for immunofluorescent staining; FSH-ß at 1:400. No staining was detected in control sections in which the first antibody was either omitted or replaced by normal mouse or rabbit serum.

The distribution of pituitary gonadotroph cells was markedly different between juvenile and adult animals. In juvenile monkeys, the vast majority of gonadotrophs was concentrated within a small region, usually near the rostral border or adjacent to the PI (Fig. 1Go, A, C, F, and G). In contrast, intensely stained gonadotrophs were observed throughout the adult pituitary gland (Fig. 1Go, B, D, E, H, and I), although, as for the immature animals, they were more densely located along the rostral border and near the PI. Compared with the isolated arrangement observed in the juvenile pituitary (Fig. 1FGo), gonadotrophs in the adult occurred as both isolated and clustered cells (Fig. 1IGo).



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  		Figure 1. Sagittal sections of the rhesus monkey pituitary gland from juvenile (A, C, F, and G) and adult (B, D, E, H, and I) animals, showing immunostained gonadotrophs using a monoclonal antibody to the LH ß-subunit. Note the different regions of the pituitary (A–E), the boundaries among the PD, PI, and pars nervosa (PN; E); the localized distribution of gonadotrophs in the juvenile (arrows, A and C); and the striking difference in gonadotroph density between juvenile (F and arrow in G) and adult (H and I) animals. PT, pars tuberalis. Magnifications: A and B, x50; C and D, x100; E, x200; F–I, x400.

 
Serum LH and testosterone concentrations

At death, serum LH concentrations were 1.9 ± 0.4 vs. 6.2 ± 1.8 ng/ml for juvenile and adult animals, respectively (P < 0.01; Fig. 2AGo), and testosterone values in the same groups were 0.8 ± 0.1 vs. 5.7 ± 1.5 ng/ml (P < 0.01; Fig. 2BGo), thus corroborating the different stage of sexual maturation between the two groups.



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  		Figure 2. Mean serum concentrations of LH (A) and testosterone (B) in juvenile (age, 1.7 ± 0.2 yr; n = 5) and adult (age, 12.3 ± 0.8 yr; n = 5) male rhesus monkeys. *, P < 0.01.

 
Developmental changes in the number and proportion of gonadotrophs in relation to all other pituitary cell types (experiment 1)

LH-gonadotrophs (i.e. LH-monohormonal and bihormonal cells) and all other cell types in the rhesus monkey pituitary gland were counted as described previously. The mean number of gonadotrophs per field increased dramatically from 22.4 ± 7.6 in the juvenile to 158.4 ± 0.14 in the adult (P < 0.01). More importantly, the proportion of LH-gonadotrophs in relation to all other pituitary cells significantly increased from 1.62 ± 0.63% in the juvenile to 10.54 ± 2.43% in the adult (Fig. 3Go; P < 0.01).



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  		Figure 3. Proportion (percentage) of gonadotrophs immunoreacted with an LH antibody in relation to the entire pituitary cell population in juvenile and adult male rhesus monkey pituitary glands. *, P < 0.01.

 
LH/FSH-gonadotroph ratio and incidence of gonadotroph subtypes as a proportion of the total gonadotroph population (experiment 2)

To determine developmental changes in the hormonal identity of the gonadotroph population, LH- and FSH-immunopositive cells (whether monohormonal or bihormonal) were studied using double immunofluorescent staining with specific antibodies in the same section. These developmental changes in the hormonal specificity of the gonadotroph population can be clearly observed in representative sections shown in Fig. 4Go. The LH/FSH-gonadotroph ratio was significantly smaller in the juvenile (0.31 ± 0.04) than the adult (1.56 ± 0.09) rhesus monkey pituitary (Fig. 5CGo; P < 0.01). Because there was no significant change in the proportion of FSH-gonadotrophs in relation to all pituitary gonadotrophs, i.e. 78 ± 11% in the juvenile, compared with 61 ± 4% in the adult (Fig. 5AGo; P = 0.20), this effect was accounted for by a significant increase in the proportion of LH-gonadotrophs, from 32 ± 9% to 90 ± 2% (Fig. 5BGo; P < 0.01). The proportion of bihormonal gonadotrophs in relation to the entire gonadotroph population also increased significantly, from 12.7 ± 4.1% in the juvenile to 61.2 ± 4.4% in the adult (Fig. 5DGo; P < 0.01).



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  		Figure 4. Double-immunofluorescent staining for LH (A and D) and FSH (B and E) in the pituitary glands of juvenile (A–C) and adult (D–F) male rhesus monkeys. The superimposed images of LH and FSH staining (C and F) allowed the identification of monohormonal and bihormonal cells, in which LH-monohormonal gonadotrophs appear red, FSH-monohormonal gonadotrophs appear green, and bihormonal gonadotrophs appear yellow. Paraffin-embedded sections were incubated with a mouse monoclonal antibody to the bovine LH ß-subunit and a rabbit polyclonal antibody to the human FSH ß-subunit. Subsequently, sections were incubated with donkey antirabbit serum conjugated to fluorescein and goat antimouse serum conjugated to rhodamine. Note the higher incidence of LH-gonadotrophs in the adult (A vs. D); the similar proportions of FSH gonadotrophs between juvenile and adult animals (B vs. E); and the dramatic reduction in FSH-monohormonal gonadotroph number (by conversion to bihormonal cells) in the adult (C vs. F). Magnification, x400.

 


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  		Figure 5. Proportion (percent) of FSH-gonadotrophs (whether monohormonal or bihormonal) (A), LH-gonadotrophs (whether monohormonal or bihormonal) (B) and bihormonal gonadotrophs (D) in relation to the total gonadotroph population, and LH/FSH gonadotroph ratio (C) in pituitary glands from juvenile and adult male rhesus monkeys. *, P < 0.01; NS, nonsignificant P > 0.05.

 
Developmental changes in the specific hormonal identity of the gonadotroph population in the primate pituitary gland (experiment 2)

Data for the incidence of LH-, FSH-, and bihormonal gonadotrophs, collected from double-immunofluorescent staining, were used to calculate the number of specific FSH-monohormonal gonadotrophs (FSH only), LH-monohormonal gonadotrophs (LH only), and bihormonal gonadotrophs (LH and FSH). In contrast to a highly significant decrease in the number of FSH-monohormonal cells, from 50.9 ± 13.5 cells/field in the juvenile to 12.4 ± 3.8 cells/field in the adult (P < 0.01), there was a dramatic increase in the number of LH-monohormonal cells, from 14.8 ± 6.7 to 49.5 ± 5.9 cells/field, for the same groups, respectively (Fig. 6Go; P < 0.01). In parallel to this change, the number of bihormonal gonadotrophs also increased significantly, from 6.7 ± 0.1 cells/field in the juvenile, to 67.1 ± 8.9 cells/field in the adult (Fig. 6Go; P < 0.01). This overt shift from a juvenile monohormonal, FSH-gonadotroph-dominated population to an adult bihormonal, LH-gonadotroph-dominated population can be observed in representative sections presented in Fig. 4Go, C and F.



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  		Figure 6. Number of FSH-monohormonal (FSH only), LH-monohormonal (LH only), and bihormonal (LH and FSH) gonadotrophs/field in pituitary glands from juvenile and adult male rhesus monkeys. *, P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of this study demonstrate that throughout sexual development there is a highly significant increase in the size of the pituitary gonadotroph population, with a concomitant shift in the proportion of the population of each hormonal subtype. These observations therefore provide a morphological basis for the differential changes in gonadotrophin secretion that normally occur during sexual maturation. The mean number of LH-secreting gonadotrophs per field increased greatly, from 22 in the juvenile to 158 in the adult. As the proportion of LH-gonadotrophs in relation to all pituitary cell types increased from the juvenile to adult by approximately 6-fold, the greater number of LH-gonadotrophs in the adult cannot simply be attributed to an increase in the size of the pituitary. The increase in GnRH secretion that encompasses pubertal development is likely to contribute to these increases through its proliferative effect on LH-gonadotroph number (30). Because high-frequency pulsatile GnRH preferentially induces synthesis of LH ß-subunit mRNA (6), this may explain the greater number of gonadotrophs synthesizing LH in detectable amounts during adulthood.

At puberty, an increase in the plasma concentrations of LH and rate of LH secretion has been well documented in primates (1, 2, 31, 32, 33, 34). This distinctive increase in LH secretion was corroborated in the current study in which the concentrations of LH in serum were approximately 3 times higher in adults than juvenile animals. To fulfill the increased requirement for LH, there must be an increase in the synthetic and secretory capacity of the existing LH-gonadotroph population and/or the size of this population. In the current study, the striking differences demonstrated between juvenile and adult animals, in both number and proportion of LH-gonadotrophs, indicate that during sexual development the capacity of the pituitary to secrete LH is raised, at least in part, by an increase in the population of LH-gonadotrophs. The reverse mechanism may be involved in modulating the capacity of the pituitary to secrete gonadotrophins in other periods during which fertility is altered (e.g. at the menopause or during seasonal anestrus in photoperiodic species). In fact, the proportion of LH-gonadotrophs in the pars tuberalis was shown to be significantly greater in sexually active than in seasonally anestrous mares (19).

This study provides conclusive evidence that all gonadotroph subtypes (i.e. LH-monohormonal, FSH-monohormonal, and bihormonal cells) are present in the pituitary gland of the rhesus monkey even before sexual maturation. The presence of these three gonadotroph subtypes has also been observed in the rodent (13, 14, 15, 16, 35), equine (19), and human (21, 22) pituitaries, whereas in the sheep some studies showed a similar arrangement (18, 36), but others reported all gonadotrophs to be bihormonal (37). Changes in the relative proportion of gonadotroph subtypes provide, at very least, a morphological basis for differential changes in gonadotrophin secretion. During pubertal development, the divergent switch from an FSH- to an LH-dominated pattern of circulating gonadotrophins may be attributed to several factors, including the increase in GnRH pulse frequency preferentially stimulating synthesis of the LH ß-subunit (6), an increase in the specific inhibition of synthesis and secretion of FSH by gonadal inhibin feedback (8), and the greater sensitivity of FSH to gonadal steroid feedback inhibition (7). However, although each of these factors may contribute by signaling the aforementioned change in gonadotrophin release, their action must ultimately manifest itself as changes at the gonadotroph level, such as in the rate of synthesis or secretion. The highly significant increase in the ratio of LH/FSH-gonadotrophs from 0.31 in the juvenile to 1.56 in the adult observed in this study, corresponds to, and is likely to be responsible for, the previously reported increase in the LH/FSH gonadotrophin ratio in plasma. Because there was no significant change in the proportion of gonadotrophs that contained FSH (in relation to all pituitary gonadotrophs), this effect was solely accounted for by the 3-fold increase in the proportion of gonadotrophs that contained LH. Furthermore, that the proportion of LH-gonadotrophs increased without a significant decrease in the proportion of FSH-gonadotrophs is indicative of an increase in the proportion of bihormonal cells. Therefore, in agreement with a previous report in the rat (27), changes in the hormonal specificity of the gonadotroph population do occur during postnatal sexual development in the primate.

Considering that each gonadotrophin (e.g. LH) can be secreted from a monohormonal (i.e. LH only) or bihormonal (i.e. LH+FSH) gonadotroph cell, a greater understanding of the changes in gonadotroph subtypes that occur at puberty was yielded by comparison of the number per unit area, rather than proportion, of exclusively LH-monohormonal, FSH-monohormonal, and bihormonal cells. Whereas the number of FSH monohormonal gonadotrophs per field dramatically decreased from the juvenile to the adult stage, there was a comparable increase in number of bihormonal gonadotrophs. Because the proportion of FSH-gonadotrophs in relation to all gonadotroph cells did not differ between juvenile and adult animals, it is likely that the greater number of bihormonal gonadotrophs in the adult was accounted for by the conversion of juvenile FSH-monohormonal gonadotrophs into bihormonal cells. The pituitary gonadotroph population may thus comprise multipotential cells, switching their hormonal identity in response to changes in the variety of signals affecting the pituitary during different physiological conditions. The idea that changes in the heterogeneity of gonadotroph cells may provide a mechanism whereby gonadotrophin secretion can be differentially regulated is supported by observations in other species. Changes in the incidence of gonadotroph subtypes have been reported throughout the annual reproductive cycle of the horse (19, 20), a species with a distinctively divergent pattern of gonadotrophins. Similarly, dynamic changes in the heterogeneity of the gonadotroph population have been identified during the estrous cycle of the rat (25, 38) and sheep (18). These cytological changes in the pattern of gonadotrophin storage not only were associated with changes in gonadotrophin secretion, but were also shown to be regulated by GnRH stimulation (39). Moreover, whereas low-frequency GnRH pulses activated the expression of FSH gonadotrophs, high-frequency GnRH increased the incidence of LH cells (26). Therefore, it is conceivable that although all gonadotrophs may be capable of synthesizing LH alone, FSH alone, or both, heterogeneity in sensitivity to the pattern of GnRH secretion could cause gonadotrophs to switch their hormonal identity through sexual maturation.

In addition to the effects of GnRH pulse frequency, there may be a role for cell surface GnRH receptor density, gonadal steroids, and paracrine interactions among different pituitary cell types in regulating heterogeneity in the gonadotroph population and thus gonadotrophin secretion. The cell-surface density of GnRH receptors has been shown to determine the level of gonadotrophin subunit expression and therefore the subtype of an individual gonadotroph; indeed, whereas optimal stimulation of LH {alpha}- and ß-subunit expression was reported to occur at relatively high cell-surface receptor densities, FSH ß-subunit expression was optimal at lower densities (40). Because GnRH pulse frequency affects the number of GnRH receptors (41, 42, 43), modulation of GnRH receptor density may be the mechanism by which GnRH pulse frequency differentially regulates the expression of LH and FSH. Furthermore, gonadal steroids have been shown to increase the number of GnRH receptors in the pituitary during both negative (44) and positive (45, 46, 47, 48) phases of feedback.

Considering that in the rat only 25% of LH-containing cells express estrogen receptor-{alpha}/ß mRNA (49) and in sheep the negative feedback of estradiol targets mostly bihormonal gonadotrophs (50), it is possible that gonadal steroids have a differential regulatory effect on gonadotroph subtypes in the primate pituitary. It was recently suggested that estrogen can cause a rearrangement of the proportions of gonadotroph subtypes through its ability to increase the number of GnRH receptors in the cell surface (50). It is noteworthy that in the present study, the concentrations of testosterone in the peripheral circulation were approximately 7 times higher in the adults than juvenile animals. Finally, it is feasible that gonadal steroid hormones affect the gonadotroph population via neighboring cells, such as lactotrophs and somatotrophs (49). Indeed, lactotrophs are known to be responsive to estradiol (51, 52) and make intimate contact with gonadotrophs in rodents (53, 54), and we recently reported that in sheep, gonadotrophs not only are completely embedded within lactotroph clusters but selectively express prolactin receptors (17). Alternatively, paracrine cell interactions may affect gonadotroph heterogeneity through a mechanism that does not involve gonadal steroids.

Taken together, the results of this study are consistent with the hypothesis that the increased LH/FSH gonadotrophin ratio in the peripheral circulation at puberty is specifically related to an increase in the number of LH-monohormonal and bihormonal gonadotrophs, with a concomitant decrease in the number of FSH-monohormonal cells. This is further supported by the observation that the proportion of bihormonal gonadotrophs in relation to all gonadotroph cells increased dramatically, from 12.7% in the juvenile to 61.2% in the adult. In conclusion, this study provides compelling morphological evidence to suggest that the secretion of the two gonadotrophins throughout sexual development can be differentially regulated by changes in the proportion of gonadotroph subtypes within the total pituitary gonadotroph population.


   Acknowledgments
 
We thank Dr. Jan F. Roser (University of California-Davis) for providing the LH antibody and Prof. Alan S. McNeilly (MRC Human Reproductive Sciences Unit, Edinburgh, UK) for the generous gift of FSH antibody.


   Footnotes
 
Abbreviations: PD, pars distalis; PI, pars intermedia; TBS, Tris-buffered saline.

Received June 27, 2002.

Accepted March 7, 2003.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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