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Regular Article |
a Department of Anatomy, University of Bristol, Bristol BS2 8EJ, England, United Kingdom
ABSTRACT
Little is known about the neuroendocrine control of fertility in the horse. In this species, unusual features characterize the normal estrous cycle such as a prolonged preovulatory LH surge during the follicular phase and a distinctive FSH surge during the midluteal phase. This study investigated the distribution and hormonal identity of gonadotrophs in the pars distalis (PD) and pars tuberalis (PT) of the equine pituitary gland as possible morphological bases for the referred unusual endocrine characteristics. In addition, the proportion of gonadotrophs in relation to other pituitary cell types during both the estrous cycle and anestrus were investigated. Pituitary glands were collected from sexually active (n = 5) and seasonally anestrous (n = 5) mares in November, and single or double immunofluorescent staining was carried out on 6-µm sections using monoclonal antibodies to the LHß or FSHß subunits and a polyclonal antibody to ovine LHß. Gonadotrophs were densely distributed around the pars intermedia in the PD and in the caudal ventral region of the PT. In addition to isolated cells, clusters of gonadotrophs were found surrounding the capillaries. No significant differences were detected in the number of gonadotrophs between sexually active and anestrous mares in either the PD or PT. In the PD, gonadotrophs represented 22.7 ± 5.8% and 19.1 ± 2.1% of the total cell density in sexually active and anestrous animals, respectively (P > 0.05). However, in the PT, gonadotrophs accounted for a higher proportion of the total cell population in sexually active (6 ± 0.1%) than in anestrous (1.2 ± 0.05%) mares (P < 0.02). Double immunofluorescence revealed that the majority of gonadotrophs were bihormonal (i.e., positive for LH and FSH); however, in the sexually active mare, a larger proportion of gonadotrophs (22.5 ± 3.6%) were monohormonal for either LH or FSH, when compared to anestrous animals (9.7 ± 1.2%; P < 0.02). Based on these findings we conclude that: 1) although the relative distribution of gonadotrophs is similar to those reported for other species, a significantly larger proportion of gonadotroph cells is present in the equine pituitary gland; 2) gonadotroph density does not appear to differ between sexually active and anestrous mares in the PD; 3) a larger proportion of gonadotrophs is apparent in the PT of sexually active animals; and 4) although a large incidence of bihormonal gonadotrophs is present in the horse, specific LH or FSH cells differentiate predominantly during the sexually active phase.
FSH, LH, seasonal reproduction
INTRODUCTION
The process of reproduction in mammals is governed by the central nervous system; information from internal (e.g., metabolic) and external (e.g., photoperiodic) cues converges in the hypothalamus, which, through the release of a single hypothalamic hormone (GnRH), regulates the synthesis and secretion of the two gonadotropins LH and FSH from gonadotroph cells in the pituitary gland [1, 2]. Secretion of GnRH is intermittent, resulting in reciprocal pulsatile gonadotropin release [3]. In the horse, however, continuous infusion of synthetic GnRH successfully stimulated continuous gonadotropin release [4]. This is apparently unique to the equine and may reflect an intrinsic difference in the hypothalamic-pituitary intercommunication, because most species require pulsatile GnRH delivery for successful gonadotropin release, otherwise desensitization occurs [4]. Preferential release of one or the other of the gonadotropins (i.e., LH/FSH) is mediated by the interplay of GnRH and gonadal secretory products [1, 5] and is accompanied by changes in the cells secreting them [6]; therefore, it has been proposed that cell composition in the pituitary should vary depending on stage of the reproductive cycle [7].
Mares are seasonally polyestrous and, like some rodents (e.g., hamsters), breed during long days. Gonadotropic patterns associated with the estrous cycle of the mare are considerably different from other species. In general, these hormones have individually distinctive profiles, with high concentrations of FSH during the luteal phase and high concentrations of LH during the follicular phase of the estrous cycle [8]. The horse, however, is a species where a marked FSH surge occurs in the midluteal phase without a simultaneous LH rise [9, 10]. It is only during the ovulatory period that concentrations of both gonadotropins increase significantly [9, 10], culminating in the gonadotropin surge that induces ovulation. In the horse, the LH surge is prolonged, beginning a few days before ovulation and continuing for several days after [11–13], contrasting sharply to the sheep that exhibits a short-lived gonadotropin surge lasting 12–24 h [14]. Although a possible explanation for this is that equine LH has a longer half-life than most species [12], morphological differences in the gonadotrophs, i.e., cell number and composition, may be of equal importance for the occurrence of the prolonged surge of gonadotropins. The estrous cycle is normally, although not always [15], interrupted during the winter months as the mare enters anestrus. If an animal is to become sexually inactive, at this time GnRH secretion is reduced dramatically and the pituitary fails to release significant amounts of FSH or LH [16]. Pituitary content of LH decreases, but FSH content does not change [16]. There is an important reduction in gonadal steroid and protein hormone secretions and lack of ovulation. Re-establishment of GnRH secretion is among the first events in the transition from anestrus to the breeding season [17] which primarily leads to an increase in FSH secretion. This GnRH increase does not result in increased secretion of LH at an early stage due to the reduced pituitary LH stores brought about in anestrus. As in other species, an increase in estrogen, prior to the LH surge that induces first ovulation, is likely to provide some stimulus within the pituitary for the synthesis and/or secretion of LH, forming part of the last chain of events of the transition to the sexually active phase [17].
An integration of the individual functions of the components of the hypothalamic-pituitary-gonadal axis is thus essential for reproductive success. Specifically for the pituitary gland, the present understanding of its function is based on experimental work conducted primarily in the rat, sheep, and monkey. Little is known, however, about the morphological and physiological characteristics of the equine pituitary. Features specific to the equine may be responsible for the aforementioned unusual endocrine characteristics of the mare's reproductive cycle. To provide initial information on the morphological basis for the differential regulation of gonadotropin secretion, and thus on the intrapituitary regulation of fertility, this study was undertaken to 1) investigate the relative distributions of gonadotrophs in the pars distalis (PD) and pars tuberalis (PT) of the equine pituitary gland, 2) determine whether the proportion of gonadotrophs in these two regions of the pituitary differ between sexually active and anestrous mares within the same season, and 3) determine whether gonadotophs are monohormonal (specific for either LH or FSH) or bihormonal (secrete both gonadotropins), and whether hormonal specificity is affected by stage of the seasonal reproductive cycle. Preliminary results from these studies have been published in abstract form [18].
MATERIALS AND METHODS
Pituitary glands from 10 mares, 5 sexually active and 5 seasonally anestrous, were collected from an abattoir immediately after death during November. Tissue collection was carried out within the same season (late autumn) in order to differentiate possible effects of reproductive stage from those of season on the pituitary cytological configuration. Gross examination of the ovaries was used to estimate cycle stage. A mare was considered to be sexually active if a recent corpus luteum and a large follicle (
2 cm) were present upon careful dissection of the gonads. In contrast, animals were said to be in anestrus when only a corpus albicans, small follicles, and no corpora lutea were present in the gonads. The pituitary glands were fixed in Bouin's for up to 46 h before immersion in 70% ethanol and then cut either transversally or sagittally. The tissues were then dehydrated in graduated alcohol and embedded in paraffin blocks. Sections (6 µm) were cut, mounted onto VECTA-coated slides, and dried overnight at 37°C. Experiments were then carried out using the following three specific antibodies: 1) bovine LHß monoclonal antibody (Mab; 518 B7—a gift from Dr. J.F. Roser, University of California, Davis); 2) ovine FSHß Mab (a gift from Dr. K. Henderson, AgResearch, Wallaceville, New Zealand); 3) ovine LHß polyclonal antibody (Pab), raised in rabbit (ASMcN R23—a gift from Prof. A.S. McNeilly, MRC Reproductive Biology Unit, Edinburgh, UK).
Each antibody was first tested through single immunofluorescent staining, to check whether it would cross-react in the horse and to determine its optimal working dilution. Tissue sections were processed following a method previously described [19]. Briefly, sections were dewaxed and rehydrated by sequential immersion in xylene and graduated alcohol and then washed in 0.05 M Tris-buffered saline (TBS, pH 7.4). Nonspecific binding sites were blocked using normal goat serum (for Mabs) or normal donkey serum (for Pab) for 1 h at room temperature (1:5 dilution in TBS). Sections were then incubated for 24 h at 4°C in a humidity chamber, with specific primary antibodies of varying dilutions (ranging from 1:50 to 1:5000). Subsequently, sections were washed in 0.01 M PBS containing 0.1% BSA (pH 7.6) and then incubated at room temperature for 1 h in secondary antibodies, i.e., goat anti-mouse serum conjugated to rhodamine (for Mabs; Sigma Chemical Co., Poole, Dorset, UK) and donkey anti-rabbit serum conjugated to fluorescein (for Pab; SAPU, Carluke, Lanarkshire, UK). Both antibodies were used at 1:20 dilutions in PBS. Finally, sections were rinsed in PBS and the coverslips mounted with Vectashield for Fluorescence (Vector Laboratories, Peterborough, UK). For control sections, primary antibodies were either omitted or replaced with normal mouse or rabbit serum.
Experiment 1
Counterstaining enabled the determination of the relative distribution and proportion of gonadotrophs in relation to other cell types in the equine pituitary. Pituitary sections were immunoreacted with LHß Mab and counterstained with hematoxylin using the following method. Sections were dewaxed and rehydrated, then washed in hydrogen peroxide (4 parts 3% H2O2:1 part methanol) for 20 min before blocking with normal rabbit serum (1:5 dilution with TBS) overnight in a humidity chamber at 4°C. The primary antibody (LHß Mab, 1:5000) was then applied for 2 h in a humidity chamber at room temperature. After rinsing in PBS/BSA for 15 min, sections were incubated with horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulin (1:200 dilution in 0.01 M PBS/1% BSA) for 1 h at room temperature. This was followed by exposure to diaminobenzidine tetrahydrochloride for up to 10 min, to visualize immunoreactivity. Counterstaining with hematoxylin provided the neutral staining of the other pituitary cell types. Sections were then washed in graduated alcohol and xylene and the coverslips mounted with DePeX before visualization under the light microscope.
Experiment 2
Double immunofluorescent staining provided the means to determine the hormonal specificity of gonadotrophs. The FSHß Mab was used in combination with the LHß Pab. The method was similar to that for single staining except when applying normal blocking sera and antibodies. Nonspecific binding sites were blocked using a combination of normal goat and donkey sera (1 part goat serum:1 part donkey serum:3 parts TBS). For application of the primary antibodies, sections were first incubated with FSHß Mab (1:100) for 20 min at room temperature, before adding LHß Pab (1:200); exposure to these primary antibodies was for 24 h at 4°C. After rinsing in PBS (2 x 5 min), secondary antibodies were applied sequentially: sections were exposed to rhodamine-conjugated goat anti-mouse serum for 1 h at room temperature (in the dark), washed in PBS (4 x 5 min), and then incubated for a further hour at room temperature with fluorescein-conjugated donkey anti-rabbit serum, before final rinsing (4 x 5 min) in PBS. As for single staining, 1:20 dilutions of the two secondary antibodies were used.
Quantitative and Statistical Analyses
Gonadotrophs, and all remaining pituitary cell types, were counted in 0.01-mm2 fields; in each counterstained section, five fields for the PD and two fields for the PT were randomly selected. Three sections, at least 500 µm apart, were analyzed from each animal and the average cell density per field for each mare calculated. Group means (for sexually active and anestrous mares) were then obtained, by combining the results of individual animals and expressed as means ± SEM. The percentage of gonadotrophs was then calculated, expressing the average density of gonadotrophs as a percentage of the average total cell density; results were expressed as the mean ± SEM percentage of the group. In addition, the relative proportions of monohormonal and bihormonal gonadotrophs were calculated. Cells positive for either LH or FSH were discriminated by their fluorescence at two wavelengths of UV light, when examined under the microscope. Double exposure photographs were taken of random fields in eight different pituitary sections (four sexually active, four seasonally anestrus). As each photo had been produced from exposure to the two different wavelengths, the resulting projection showed all gonadotrophs, the hormone identity of which could be determined by their color. The total numbers of LH- and FSH-monohormonal cells were counted from each photo and expressed as a percentage of the total number of gonadotrophs in that photo. Final results were calculated as percent mean ± SEM of the group.
The effects of reproductive state on the number and proportion of gonadotrophs in the equine pituitary gland and on the percentage of monohormonal cells were examined by ANOVA, using the StatView computer program. Significance was set at P < 0.05.
RESULTS
All antibodies used were found to cross-react in the horse. For immunofluorescent staining, the LH monoclonal antibody positively reacted at all dilutions tested. In comparison, the polyclonal LH antibody only reacted at higher concentrations. The following concentrations were used in subsequent experiments: LHß Mab: 1:1000 dilution; FSHß Mab: 1:100 dilution; LHß Pab: 1:200 dilution. For nonimmunofluorescent staining, the 1:1000 dilution of LHß Mab gave an extremely high background and therefore dilutions of 1:1500, 1:2000, 1:4000, and 1:5000 were sequentially tested; it was evident that the 1:5000 dilution provided the best results. No staining was detected in control sections, where the first antibody was omitted or replaced by normal mouse or rabbit serum, during any of the immunofluorescent or nonimmunofluorescent runs (Fig. 3, L and P).
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Experiment 1
Distribution of gonadotrophs in the equine pituitary gland The different regions of the equine pituitary gland are shown in Figure 1A. Gonadotrophs were found throughout the PD and PT, with no specific immunoreactive LH staining in the pars nervosa (Fig. 1B). In the PD, denser populations of gonadotrophs were observed surrounding the pars intermedia and the portal vessels (Fig. 1C), while in the PT, large numbers of gonadotrophs were found in the caudal ventral region (Fig. 1D). The rostral and dorsal regions of the PT had a reduced population of gonadotrophs.
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Number and proportion of gonadotrophs, in relation to other cell types, in the PD and PT of the equine pituitary gland No significant differences were seen in the number or proportion of gonadotrophs in the PD of sexually active compared to anestrous animals, with gonadotrophs representing 22.7 ± 5.8% and 19.1 ± 2.1% of the total pituitary cell population, respectively (Fig. 2). In contrast, there was a tendency for a greater gonadotroph number in the PT of sexually active compared to anestrous mares, although the difference did not reach statistical significance (P = 0.1). However, the proportion of gonadotrophs in the PT differed between the two stages of the reproductive cycle, with a significantly higher (P < 0.02) incidence of gonadotroph cells in cyclic mares. In this region, gonadotrophs accounted for 6 ± 0.1% of the pituitary cell population in sexually active mares compared to 1.2 ± 0.05% in anestrous animals (Fig. 2).
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Experiment 2
Hormonal specificity of gonadotrophs in the PD and PT of the equine pituitary gland Populations of both bihormonal and specific LH or FSH monohormonal gonadotrophs were found in both the PD (Fig. 3, A through C and Fig. 3, E through G) and the PT (Fig. 3, I through K and Fig. 3, M through O) of sexually active and anestrous mares, although bihormonal (i.e., LH/FSH) cells accounted for the majority of gonadotrophs (Fig. 3, C, G, K, and O). However, the proportion of monohormonal cells detected in sexually active mares (22.5 ± 3.6%; Fig. 3D) was significantly higher (P < 0.02) than that observed for anestrous animals (9.7 ± 1.2%; Fig. 3H). These proportions are evident when simultaneous exposure to double immunofluorescent staining was carried out for the immediate identification of the cell type, with bihormonal gonadotrophs represented by yellow cells, monohormonal LH gonadotrophs by green cells, and monohormonal FSH gonadotrophs by red cells (Fig. 3, D and H).
DISCUSSION
Previous work has shown that the different cell types of the pituitary are not randomly distributed in the species so far studied [20]. The results presented here agree with those findings, with specific arrangements observed supporting reports for the rat [20–23], sheep [19, 24, 25], and other large mammals [26]. Specifically, in the PD, although gonadotrophs were found distributed throughout the region, denser populations extended along either side of the pars intermedia and near the infundibulum attachment. Gonadotrophs were also found in the medial region of the PD that is adjacent to the PT. In the PT, gonadotrophs were concentrated in the caudal ventral region with very few cells in the rostral and dorsal regions. Although it was originally suggested that PT cells are similar to those of the PD [22], reports from other species suggest gonadotrophs do not have common properties in these regions. Studies in the rat, for example, indicate that the mode of hormone synthesis and/or release from LH cells of the PT is different from those of the PD [24, 27]. The secreting capacity of these cells appeared to be small and inconsistent, with low GnRH sensitivity [27]. This may imply a less important endocrinological role for the PT in this species. In contrast, in sheep, LH cells are larger and have a higher hormone content in the PT than in the PD [24], suggesting that in this seasonal breeder the rate of hormone synthesis is higher in the PT.
In both the PD and PT of the equine pituitary, gonadotrophs were arranged alongside the portal blood vessels. Such an arrangement implies an important functional relationship to the pituitary blood supply and a control point for hypothalamic regulation. The relationship should be more apparent in the PT, being in closer contact with the nerve endings of the median eminence of the hypothalamus, that are themselves in close contact with the portal vessels [28]. As gonadotrophs were concentrated in the caudal ventral region of the PT where it merges with the PD, this spatial relation suggests that secretory products of the PT may modulate hypothalamic effects on the PD. Moreover, these cells are likely to play an important role in the short-loop feedback regulation of gonadotropins on their own secretion [29]. This mechanism is likely to operate due to the close association of those gonadotrophs with the median eminence. Further interactions between cells may exist because gonadotrophs were found as both isolated and clustered populations, supporting observations in other species [rat: 21, 30; sheep: 24]. A clustered arrangement may provide the means for self-regulation between neighboring gonadotrophs. In contrast, isolated cells may be regulated by other pituitary cell types, as proposed for rodents [31] and sheep [19].
A larger proportion of gonadotrophs (ranging from 13% to 38%) were present in the equine pituitary compared to other species (rat: 14% [32]; sheep: 13% [33]). However, within the PD, there were no significant differences in this study in the average densities of gonadotrophs between sexually active and anestrous animals. In the PT, the mean number of gonadotrophs tended to be higher in sexually active compared to anestrous mares, but the difference in numbers did not reach statistical significance, perhaps as a result of the reduced sample size. However, in the PT the proportion of gonadotrophs in relation to other pituitary cell types was significantly higher in sexually active animals, with the gonadotrophs accounting for 6% of the total cell population compared to 1.2% in anestrous mares. In the sheep, gonadotrophs represented less than 1% in corresponding regions of the PT [25], and no differences in the density of gonadotrophs in relation to season were found in this species [25]. Results of the present study suggest that there may be a change in the gonadotroph cells of the PT in relation to stage of the reproductive cycle, because a marked tendency for a decrease in the number of gonadotrophs was observed in anestrus. Interestingly, the total number of other pituitary cells in the region increased. As a result of these two changes (decreased number of gonadotrophs and increased total number of other cell types), the gonadotrophs represented a significantly smaller proportion of the cell population in anestrus. Because the pituitary glands of both groups of animals in the current study were collected in November, these findings may reflect a direct effect of reproductive state rather than an effect of season. Differences in sensitivity to photoperiodic effects cannot be completely ruled out, however; indeed it has been reported that thyrotroph density may decrease in response to inhibitory photoperiod [34], supporting the idea that the proportions of a specific cell type may be affected by day length. Such changes are likely to be melatonin induced since, in sheep, high-density melatonin-binding sites are concentrated in the PT [35, 36]. With the additional observation of high concentrations of estrogen receptors expressed in the ovine PT [37], this tissue may play an important role in the control of gonadotropin secretion, where the effects of estrogen and melatonin may be integrated to modulate reproduction.
This study has provided conclusive evidence that the equine pituitary contains populations of gonadotrophs that store either LH or FSH (i.e., monohormonal) or both gonadotropins (i.e., bihormonal). Previous studies have shown that gonadotrophs display a similar arrangement in rodents [23, 38–40]. Controversy still exists regarding the sheep, where some authors reported a similar situation to that of rodents [24], but others have shown that all gonadotrophs contain both hormones [41]. In the current study, bihormonal and monohormonal cells were distributed throughout the anterior pituitary of the mare; this differs with reports in other species where peripheral cells were shown to secrete one gonadotropin and central cells the other [20, 42]. It is important to note that in both, sexually active and anestrous mares, the majority of gonadotrophs were bihormonal. However, whereas monohormonal cells accounted for 22.5% of the total gonadotroph density in sexually active mares, only 9.7% were detected in anestrous animals. These results suggest a shift from monohormonal gonadotrophs to bihormonal cells during anestrus, although it remains possible that some cells become undetectable due to reduced gonadotropin content at this stage. The decrease in monohormonal cells during this period could explain, at least in part, the reduction in pituitary LH content observed during this period [16]. The cellular functional heterogeneity observed in the current study may represent a morphological basis for the differential release of gonadotropins that occurs during the reproductive cycle and implies that the amount of gonadotropin released will depend on which gonadotrophs are stimulated. The idea of morphologically and functionally distinct subpopulations of gonadotrophs was first suggested by Denef et al. [30, 38, 43] and forms a basis for the selective stimulation of gonadotropin release by a single hypothalamic factor. However, not only could changes in hormone secretion further be brought about by the specific pattern of GnRH release seen in the mare during the reproductive cycle, but biochemical changes in the cell may also contribute [44]. In the rat, GnRH shifts the proportion of cells to mainly bihormonal by enlargement [45]; the large gonadotrophs are stimulated to secrete FSH (but not LH) at low GnRH concentrations, whereas smaller cells only respond at higher GnRH concentrations [43]. The intracellular basis for the different release is unknown. What is generally accepted, however, is that the gonadotropins are stored and secreted from different secretory granules within bihormonal cells [41, 46], which could account for the differential release. The foregoing hypotheses imply that the pituitary itself may be the site for differential release of gonadotropins, and characteristics intrinsic to the gonadotroph cell population should be considered as a basis for the differential response to a single releasing hormone.
In summary, this study has shown that 1) although the relative distribution of gonadotroph cells is similar to that of other species, a larger incidence of gonadotrophs is present in the equine pituitary; 2) in the PD, gonadotroph density does not appear to differ between sexually active and anestrous mares; 3) a larger proportion of gonadotroph cells is apparent in the PT of sexually active animals; and 4) although a large incidence of bihormonal gonadotrophs is found in the horse, specific LH or FSH cells appear to differentiate predominantly during the sexually active phase. This study therefore provides morphological bases for the differential regulation of gonadotropin secretion and for a role of the PT in seasonal breeding, in a species that shows dramatic changes in fertility throughout the annual reproductive cycle.
ACKNOWLEDGMENTS
We thank Dr. J.F. Roser and Quidel Corporation (San Diego, CA) for the gift of LHß Mab; Dr. K.M. Henderson for the FSHß Mab, and Prof. A.S. McNeilly for the LHß Pab. We also thank Christine Goodall for her assistance in immunofluorescent runs and technical support throughout the study, Susan Gregory for her contributions to the art work, and Potters Abattoir (Cappards Farm, Bishop Sutton, Bristol, UK) for providing equine specimens.
FOOTNOTES
First decision: 30 November 1999.
1 Correspondence: Domingo J. Tortonese, Dept. of Anatomy, University of Bristol, Southwell St., Bristol BS2 8EJ, England, UK. FAX: 44 117 925 4794. 
Accepted: April 17, 2000.
Received: October 21, 1999.
REFERENCES
This article has been cited by other articles:
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  D. Meeran, H. F. Urbanski, S. J. Gregory, J. Townsend, and D. J. Tortonese Developmental Changes in the Hormonal Identity of Gonadotroph Cells in the Rhesus Monkey Pituitary Gland J. Clin. Endocrinol. Metab., June 1, 2003; 88(6): 2934 - 2942. [Abstract] [Full Text] [PDF]
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