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Pituitary |
Department of Anatomy,2 University of Bristol, Bristol BS2 8EJ, England, United Kingdom
Medical Research Council Human Reproductive Sciences Unit,3 Edinburgh EH16 4SB, Scotland, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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dopamine, luteinizing hormone, pituitary, prolactin, seasonal reproduction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A specific morphological arrangement between gonadotrophs and lactotrophs has provided evidence for an interaction between these endocrine cells in the pars distalis of the rat pituitary. Clustered lactotrophs were frequently observed to surround isolated gonadotrophs [13, 14], and a paracrine communication was identified using a fluorescent molecule that could pass freely through gap-junction channels between adjacent cells [15]. Substantial evidence therefore exists for a direct effect of PRL on gonadotropin release in rodents. However, possible effects of PRL on LH release in other species are not as well characterized. In sheep, evidence that PRL may act directly within the pituitary gland emerged from the observation that gonadotrophs of the ewe were also completely surrounded by lactotroph cell clusters and were identified as the only pituitary cells that contain PRL receptors (mRNA and protein) [16]. The close gonadotroph-lactotroph association observed in the pars distalis of the pituitary of this species along with the presence of PRL receptors within gonadotrophs are suggestive of the existence of a mechanism by which PRL may regulate gonadotropin secretion through a paracrine interaction. These possible actions of PRL on gonadotropin secretion, directly within the pituitary gland, may prove to be vital in our understanding of the central control of fertility in a species that displays a clear, inverse correlation between the secretion of these two hormones throughout its annual reproductive cycle.
In the present study, the effects of PRL on the LH response to GnRH were investigated in ovine pituitary primary cell cultures during the seasonal reproductive cycle in an attempt to elucidate whether PRL is involved in the intrapituitary regulation of gonadotropin secretion.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pituitary glands from female sheep were obtained during the winter month of January (breeding season [BS]) and the summer months of June and July (NBS) from animals killed in an abattoir (n = 20/season). The reproductive status of the animals (i.e., whether sexually active or inactive) was confirmed by gross examination of the ovaries for the presence or absence of corpora lutea. Pituitaries were dissected from surrounding tissue soon after death and used for primary cell cultures according to a method previously described [17]. Briefly, the tissue was washed in incomplete medium M199 (Life Technologies, Paisley, Scotland, UK), and the pars nervosa was removed from the dorsal aspect of the pituitary and discarded. The pars distalis and pars tuberalis were dispersed by incubation with a 0.1% collagenase D (Boehringer Mannheim, Mannheim, Germany) and hyaluronidase (Sigma-Aldridge Ltd., Poole, Dorset, England, UK) solution in a shaking water bath at 37°C for 75 min. The tissue was then removed from the enzymatic solution and dispersed manually in Hanks buffered salt solution (Sigma-Aldridge) containing 2 mM EDTA (pH 7; BDH Laboratory Supplies, Poole, Dorset, England, UK). The dispersed cells were subsequently mixed with complete medium M199 (Life Technologies) containing 10 mg/ml of insulin, 50 mg/ml of gentamicin, 100 IU/ml of penicillin-streptomycin (all reagents from Sigma-Aldridge), and 10% steroid-free lamb serum (Life Technologies). The mixed pituitary cells were resuspended in complete medium M199 and plated at a density of 400 000 cells/well in 24-well plates.
Experimental Design
During both the BS and NBS experiments, cells were maintained in culture for 7 days, and the medium was changed every 2 days. Previous studies have demonstrated the validity of this method for producing a reliable gonadotropin dose response to exogenous GnRH in vitro [17, 18]. To investigate the effect of PRL on the GnRH-induced LH release, the wells were divided into seven experimental groups as follows: 1) control, 2) acute 2-bromo-
-ergocryptine (bromocriptine; ABr), 3) chronic bromocriptine (CBr), 4) ABr plus exogenous rat PRL (ABr+PRL), 5) CBr plus exogenous rat PRL (CBr+PRL), 6) exogenous rat PRL (PRL), and 7) TRH. During the 7-day culture period, bromocriptine treatment was applied to the CBr and CBr+PRL groups. All other experimental groups remained untreated for this period. On Culture Day 7, treatments were applied according to their designated experimental group and were immediately followed by the application of GnRH at one of the following concentrations: 0, 10–10, 10–9, 10–8, or 10–7 M. For each experimental group, three wells were assigned per dose of GnRH. Bromocriptine (a specific DA-D2-receptor agonist; Sigma-Aldridge) was administered at a concentration of 10–8 M to reduce the endogenous PRL within the cell culture media; this dose of bromocriptine has been shown previously to effectively suppress PRL secretion from ovine pituitary cell cultures [17]. To increase the concentration of PRL in the culture media, exogenous rat PRL was administered at a concentration of 500 ng/ml (rat PRL lot AFP- 6452B; National Institute of Diabetes and Digestive and Kidney Diseases [NIDDK], Bethesda, MD). Because the biological actions of PRL are not species specific, this rat PRL preparation is expected to activate ovine PRL receptors and to induce a physiological response (A.F. Parlow, personal communication). In addition, endogenous PRL secretion by lactotroph cells within the culture was stimulated by application of 10–7 M TRH (Sigma-Aldridge). To ensure that the administration of bromocriptine itself was not resulting in any effect on gonadotropin release that was unrelated to the suppression of PRL secretion, bromocriptine was administered, both acutely and chronically, in combination with exogenous rat PRL. The use of a hormone from a different species was to differentiate immunologically the endogenous from the exogenous source.
Cells were incubated for a 90-min period at 37°C following application of the above treatments and GnRH. The medium was then removed from the wells, and RIA was conducted to quantify the concentrations of LH and PRL. The experiments were repeated three times each during the BS and NBS.
Hormone Assays
The LH and PRL concentrations were determined using previously validated RIAs [19, 20]. Concentrations of LH were measured using iodinated ovine LH (LER-1056-C2) as tracer. The LH standard (NIH-LHs 23; NIDDK) was used over a range of 0.1–50 ng/ml. The ovine LH antibody (ASMN R 29) was used at a working dilution of 1:120 000. The limit of detection for the assay (90% B/Bo) was 0.2 ng/ml, and the mean intra- and interassay coefficients of variation, based on low-, medium-, and high-quality control samples, were less than 16%.
To determine whether the endogenous PRL concentrations within the culture environment were affected by the bromocriptine or TRH treatments, ovine PRL concentrations were determined according to the method of McNeilly and Andrews [19]. The limit of detection for the assay, expressed as NIH-PRLs 15 (NIDDK), was 0.5 ng/ml. The mean intraassay coefficient of variation was 6.7%. The concentration of exogenous PRL in the culture wells where rat PRL had been administered was determined using NIDDK-rPRL-1-6 as iodinated antigen, NIDDK-rPRL-RP-3 as standard, and NIDDK-anti-rPRL-S-9 as antiserum at a concentration of 1: 87 500. The intraassay coefficient of variation was 10.1%. No cross-immunoreactivity occurs in these two PRL RIAs for the opposite species; that is, rat PRL does not cross-react in the ovine PRL assay (A.S. McNeilly, unpublished data), and vice versa (A.F. Parlow, personal communication).
Statistical Analyses
In both the BS and NBS cultures, a total of seven separate experimental groups were treated with GnRH. For each group, three wells were assigned to each dose of GnRH, and the experiments were carried out three times. No differences among replicates were observed. The reported values in the present study represent the mean ± SEM. The effects of experimental treatment, dose of GnRH, and season on the secretion of LH from ovine pituitary primary cell cultures were examined by a three-way ANOVA. Because of a statistically significant three-factor interaction, two-way ANOVA was then used to examine the effects of experimental group and dose of GnRH on LH secretion individually for each season. To determine whether the GnRH-induced LH response differed in the experimental treatment groups with respect to the control, pairwise comparisons were made within each season.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The effects of treatments on the mean concentrations of LH following stimulation with GnRH in pituitary cell cultures from the NBS (i.e., summer) are depicted in Figure 1. The control group, in which no additional treatment was added, showed a dose response in LH release following the administration of increasing doses of GnRH from 0 to 10–7 M. At this time of the year, basal values of LH of 3.01 ± 0.44 ng/ml were recorded in the absence of GnRH stimulation. Administration of GnRH induced maximal LH release at a dose of 10–9 M, at which a fourfold increase (14.48 ± 1.00 ng/ml) above basal values was detected. No significant effects on the LH response to GnRH could be observed by the administration of ABr, CBr, PRL alone, or TRH. In contrast to these observations, the application of PRL in combination with bromocriptine, either acutely or chronically, resulted in a highly significant suppression of the LH response to GnRH at all doses tested (response to 10–9 M GnRH: 14.48 ± 1.0, 3.0 ± 0.09, and 3.61 ± 0.25 ng/ml for control, ABr+PRL, and CBr+PRL, respectively; P < 0.01). Even at a pharmacological dose of 10–7 M, GnRH was unable to evoke an LH response from the gonadotroph cells following treatments with ABr+PRL or CBr+PRL.
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Effects of Treatments on LH Response to GnRH in Ovine Pituitary Primary Cell Cultures During the BS
The effects of treatments on the mean concentrations of LH in the media from ovine pituitary primary cell cultures from the BS (i.e., winter) are shown in Figure 2. A clear dose response to increasing concentrations of GnRH can be observed in the control group. Basal LH values of 2.00 ± 0.13 ng/ml were detected at this time of the year, with a sevenfold increase to 15.64 ± 0.91 ng/ml in response to 10–9 M GnRH. Maximal LH release was greater in the BS than in the NBS (20.04 ± 0.22 vs. 14.50 ± 1.00 ng/ml for control groups of BS and NBS, respectively). As for the NBS, submaximal stimulation of LH release was observed at the greatest dose of GnRH (10–7 M). In contrast to the results observed for the NBS, no statistically significant effect on the LH response to GnRH could be detected for any of the experimental treatments in the BS.
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Endogenous PRL Response to Bromocriptine and TRHin Ovine Pituitary Primary Cell Cultures
The endogenous PRL concentrations in the control, bromocriptine, and TRH groups were examined during both the BS and NBS (Fig. 3). In the controls, basal ovine PRL concentrations of 238.81 ± 7.44 and 268.29 ± 14.02 ng/ ml were recorded for the NBS and the BS, respectively. Application of CBr significantly (P < 0.01) suppressed the basal concentrations of PRL during the BS (101.86 ± 3.29 ng/ml). During the NBS, a reduction in the PRL concentration was also observed (182.85 ± 9.30 ng/ml), although this did not reach statistical significance (P > 0.01). Conversely, treatment with TRH dramatically stimulated endogenous PRL release to 558.75 ± 33.57 and 465.24 ± 38.47 ng/ml during the NBS and the BS, respectively (P < 0.01). Thus, ovine PRL concentrations following the application of TRH increased to 134% and 73% above basal values at these two stages of the annual reproductive cycle.
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Exogenous PRL Concentrations in Ovine Pituitary Primary Cell Cultures
PRL concentrations were significantly increased in wells receiving exogenous rat PRL (P < 0.01). An average value of 616 ± 81.09 ng/ml was recorded after rat PRL was applied. Thus, the concentrations of exogenous (rat) PRL in this experimental group were within the same range as those of endogenous (ovine) PRL in the TRH-treated group.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During both the BS and NBS, LH release in controls displayed a classical dose response to increasing concentrations of GnRH. Maximal LH values during the BS were greater than those recorded during the NBS. This observation is in agreement with the reported decrease in circulating gonadotropin concentrations present in vivo during the NBS in sheep [1, 2, 21]. Several factors, including the decreased storage of LH granules in gonadotroph cells of pituitaries harvested during the NBS and/or alterations in the number of GnRH receptors present on the cell membrane, may contribute to the reduction in LH release at this stage. As previously shown in rats [22–24], the frequency of GnRH pulses can also modulate the amount of LHß mRNA in the ewe, with greater frequencies stimulating a greater increase in LHß mRNA than lesser frequencies do [25]. Because GnRH pulse frequency is reduced during the NBS, when sheep are sexually inactive [26, 27], a concomitant decrease in both LHß mRNA and GnRH-receptor number could be responsible for the reduced LH response of gonadotrophs in cultures from this stage of the annual reproductive cycle. The LH response to pharmacological doses of GnRH (10–8 and 10–7 M) was less than that observed at physiological concentrations (10–9 M). In fact, GnRH hyperstimulation has been shown to result in desensitization of the GnRH receptor by increasing both GnRH- receptor microaggregation and internalization [28], with subsequent down-regulation of the LHß subunit mRNA [29]. It is possible that both these mechanisms may have contributed to the reduction in LH release observed in the present study following maximal doses of GnRH.
A clear suppression of LH secretion by PRL has been reported previously. In vivo, hyperprolactinemia has been shown to cause a significant reduction of LH release [4, 5, 30] and to result in gonadal inactivity [31]. In vitro studies with rats have demonstrated a suppressive effect of PRL on the gonadotropic axis by a direct action on the pituitary [10]; whereas both basal and GnRH-stimulated LH release were inhibited by exogenous PRL, bromocriptine enhanced the stimulatory effects of GnRH. However, in the present study, bromocriptine or PRL treatments alone did not affect the LH response to GnRH during either the BS or the NBS. No statistically significant effect was observed as a result of administration of either CBr or ABr treatments. Furthermore, no difference in either basal or GnRH-stimulated LH release was detected at any of the GnRH doses employed. The application of PRL itself was predicted to act as a suppressor of LH release, but no significant effect was detected following exogenous PRL, or TRH-induced increase in endogenous PRL, within the culture. The absence of any significant effect of these treatments was somewhat unexpected. However, some evidence from previous studies indicates that the sensitivity of the LH axis to PRL may be reduced in ewes compared with other species. In nonpregnant red deer, chronic treatment with bromocriptine treatment, to suppress the increase in PRL that is associated with the onset of seasonal anestrus, significantly delayed termination of the BS [32]. In contrast, in ewes, chronic treatment with bromocriptine treatment did not affect the duration of the BS, despite significantly suppressing PRL concentrations [33].
The most striking result of the present study was observed during the NBS, when bromocriptine and PRL were applied in combination. Administration of PRL in conjunction with either ABr or CBr treatments resulted in a complete suppression of LH release at all doses of GnRH. This interaction between the PRL and dopamine axes directly within the pituitary gland to regulate LH release is, to our knowledge, a novel observation. Although the exact mechanisms by which this effect is exerted are unknown, at least two explanations can be proposed.
First, strong evidence suggests a paracrine interaction between the cells of the pituitary gland. Specific interactions between gonadotrophs and lactotrophs have been reported in immunocytochemical studies with rats, sheep, and horses [14, 16, 34], and PRL-receptor mRNA and protein have been shown to be selectively localized in the gonadotroph cells of sheep [16]. In addition, rat pituitary cells superfused with GnRH displayed a sustained increase in PRL when lactotrophs were coaggregated with an enriched population of gonadotrophs [35]. In the present study, close cell-to-cell associations were maintained in the culture system used, despite the dispersed nature of its preparation, and a significant increase in PRL release was observed following GnRH application (data not shown). The existence of close interactions between gonadotroph and lactotroph cells may facilitate the inhibition of LH release in response to bromocriptine plus PRL. Dopamine negatively regulates basal PRL secretion via activation of D2-receptors located in the lactotroph cell membrane [36]. However, dopamine binding to the lactotroph may also trigger an intercellular signal between gonadotroph-lactotroph communications, thus enabling PRL to inhibit the GnRH stimulation of LH release during the NBS. The seasonal component of this response may be the result of changes in gap-junction cell number throughout the reproductive cycle, a phenomenon detected in mink [37].
Second, an alternative mechanism may reside within the gonadotroph itself. Dopamine-binding sites have been detected in the pituitary gland of rats [38], and dopamine receptors have been reported to be present in immortalized gonadotroph cell lines, in which dopamine suppressed levels of the common gonadotropin subunit mRNA [39]. In sheep, the inhibitory effects of dopamine on LH secretion are well recognized and are mediated by D2-receptors in both males and females [40, 41]. Although most evidence suggests that the major role of dopamine is in the mediobasal hypothalamus to suppress GnRH output [42–44], dopamine also appears to affect LH secretion directly within the pituitary in this species. Indeed, dopamine was shown to inhibit the LH response to exogenous GnRH in pituitary stalk-transected ewes [45]. Whether dopamine and PRL have a synergistic role in the inhibition of gonadotropin secretion directly in the gonadotroph cell is unknown, but it appears that these two hormones may interact intracellularly to contribute to the regulation of gonadotropin secretion in sheep. The presence of GnRH receptors, PRL receptors, and dopamine receptors in a single cell provides the basis for a complex interaction that may prove to be vital in our understanding of the rapid and complete suppression of the LH response to GnRH observed in cultured gonadotroph cells from the NBS following combined treatment with bromocriptine plus PRL. GnRH, PRL, and bromocriptine exert their effects on cells of the pituitary via separate intracellular signaling pathways. GnRH binds to its G protein-coupled receptor to stimulate phospholipase C production and the subsequent formation of 1,4,5-triphosphate (IP3) and diacylglycerol [46, 47]. The increase in IP3 concentrations activates the release of calcium from intracellular stores, which in turn stimulates gonadotropin release [48, 49]. Unlike the calcium-dependent release of LH, PRL receptors function through JAK2 and STAT5 second-messenger systems [50], whereas dopamine reduces adenylate cyclase activity to inhibit the production of cAMP and IP3 [51, 52]. An interaction of these second- messenger systems may have a crucial role in inhibiting gonadotropin release in response to simultaneous administration of bromocriptine plus PRL. In particular, the suppression of IP3 by dopamine directly within the gonadotroph cell may inhibit LH release in sheep when PRL-receptor signaling pathways are also activated.
In contrast to the results observed during the NBS, no significant effect on the LH response to GnRH could be detected for any treatment during the BS. These results indicate not only an interaction between the dopaminergic and PRL systems in the regulation of gonadotropin secretion but also a seasonal/photoperiodic modulation of this mechanism. The photoperiodic component underlying alterations in this mechanism likely operates through the pineal hormone melatonin [53, 54]. It is generally agreed that the effects of photoperiod/melatonin on the reproductive axis in sheep are mediated by hypothalamic networks that impinge on GnRH neurons [55, 56]. However, melatonin receptors are also present within the pars tuberalis and the zona tuberalis of the ovine pituitary gland [57]. Therefore, melatonin is able to transfer photoperiodic information directly to the cells of the pituitary and, indeed, is reported to modulate the GnRH-induced LH release from pars tuberalis explants [58]. Moreover, in hypothalamopituitary-disconnected sheep, melatonin has been shown to suppress PRL secretion from the pars distalis by acting directly in the pituitary [59], probably through a paracrine mechanism after binding to its receptors in the pars tuberalis. Although seasonal changes in the amounts of PRL-receptor mRNA have not been detected throughout the ovine annual reproductive cycle [16], evidence suggests that GnRH-receptor and dopamine-receptor number may be regulated by changes in GnRH pulse frequency and steroidal environment, respectively [23, 24, 60]. Therefore, seasonal variations in the LH response to bromocriptine and PRL may be mediated by alterations in GnRH-receptor number and/or the differential regulation of dopamine-receptor subtype.
In conclusion, the results of the present study show that PRL alone is unlikely to modify the gonadotropin response to GnRH in sheep. However, the present study also provides evidence for an interaction between PRL and dopamine in the regulation of LH secretion directly within the pituitary gland. The exact mechanism by which these two hormones exert a synergistic inhibitory effect on the GnRH-induced gonadotropin release is unknown, but a photoperiodic modulation of this process is clearly apparent, with gonadotroph cells from the NBS being much more responsive to this interaction than those from the BS. These results prompt us to suggest that either an intercellular interaction between the gonadotroph and lactotroph cells is present or an intracellular association between two independent signaling pathways is activated to induce the dramatic and complete suppression of the LH response to GnRH that was observed during the sexually inactive phase of the annual reproductive cycle.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received: 1 September 2003.
First decision: 29 September 2003.
Accepted: 16 December 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit gene expression by dopamine D2-receptor agonist in clonal mouse gonadotroph T3-1 cells. Biol Reprod 2002 67:1218-1224This article has been cited by other articles:
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  H. L Henderson, J. Townsend, and D. J Tortonese Direct effects of prolactin and dopamine on the gonadotroph response to GnRH J. Endocrinol., May 1, 2008; 197(2): 343 - 350. [Abstract] [Full Text] [PDF]
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 H. L Henderson, D. J Hodson, S. J Gregory, J. Townsend, and D. J Tortonese Gonadotropin-Releasing Hormone Stimulates Prolactin Release from Lactotrophs in Photoperiodic Species Through a Gonadotropin-Independent Mechanism Biol Reprod, February 1, 2008; 78(2): 370 - 377. [Abstract] [Full Text] [PDF]
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 V. L. Williams, A. DeGuzman, H. Dang, M. Kawaminami, T. W. C. Ho, D. G. Carter, and A. M. Walker Common and specific effects of the two major forms of prolactin in the rat testis Am J Physiol Endocrinol Metab, December 1, 2007; 293(6): E1795 - E1803. [Abstract] [Full Text] [PDF]
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C. Brancia, P. Nicolussi, P. Cappai, G. La Corte, R. Possenti, and G.-L. Ferri Differential expression and seasonal modulation of VGF peptides in sheep pituitary J. Endocrinol., July 1, 2005; 186(1): 97 - 107. [Abstract] [Full Text] [PDF]
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