Photoperiodic modulation of the suppressive actions of prolactin and dopamine on the pituitary gonadotropin responses to gonadotropin-releasing hormone in sheep.

Abstract

In a variety of species, the LH-secretory response to gonadotropin-releasing hormone (GnRH) is completely suppressed by the combined actions of prolactin (PRL) and dopamine (DA). In sheep, this effect is only observed under long days (nonbreeding season [NBS]). To investigate the level at which these mechanisms operate, we assessed the effects of PRL and bromocriptine (Br), a DA agonist, on the gonadotropin-secretory and mRNA responses to GnRH in pituitary cell cultures throughout the ovine annual reproductive cycle. As expected, the LH-secretory response to GnRH was only abolished during the NBS following combined PRL and Br application. Conversely, the LHB subunit response to GnRH was reduced during both the BS and NBS by the combined treatment and Br alone. Similar results were obtained in pars distalis-only cultures, indicating that the effects are pars tuberalis (PT)- independent. Further signaling studies revealed that PRL and Br alter the LH response to GnRH via convergence at the level of PLC and PKC. Results for FSH generally reflected those for LH, except during the BS where removal of the PT allowed PRL and Br to suppress the FSH-secretory response to GnRH. These data show that suppression of the LH-secretory response to GnRH by PRL and DA is accompanied by changes in mRNA synthesis, and that the photoperiodic modulation of this inhibition operates primarily at the level of LH release through alterations in PKC and PLC. Furthermore, the suppressive effects of PRL and DA on the secretion of FSH are photoperiodically regulated in a PT-dependent manner.

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BIOLOGY OF REPRODUCTION (2012) 86(4):122, 1–9
Published online before print 1 February 2012.
DOI 10.1095/biolreprod.111.096909
Photoperiodic Modulation of the Suppressive Actions of Prolactin and Dopamine on
the Pituitary Gonadotropin Responses to Gonadotropin-Releasing Hormone in Sheep1
David J. Hodson,3Helen L. Henderson, Julie Townsend, and Domingo J. Tortonese2
Department of Anatomy, University of Bristol, Bristol, England, United Kingdom
ABSTRACT
In a variety of species, the LH-secretory response to
gonadotropin-releasing hormone (GnRH) is completely sup-
pressed by the combined actions of prolactin (PRL) and
dopamine (DA). In sheep, this effect is only observed under
long days (nonbreeding season [NBS]). To investigate the level at
which these mechanisms operate, we assessed the effects of PRL
and bromocriptine (Br), a DA agonist, on the gonadotropin-
secretory and mRNA responses to GnRH in pituitary cell
cultures throughout the ovine annual reproductive cycle. As
expected, the LH-secretory response to GnRH was only
abolished during the NBS following combined PRL and Br
application. Conversely, the LHB subunit response to GnRH was
reduced during both the BS and NBS by the combined treatment
and Br alone. Similar results were obtained in pars distalis-only
cultures, indicating that the effects are pars tuberalis (PT)-
independent. Further signaling studies revealed that PRL and Br
alter the LH response to GnRH via convergence at the level of
PLC and PKC. Results for FSH generally reflected those for LH,
except during the BS where removal of the PT allowed PRL and
Br to suppress the FSH-secretory response to GnRH. These data
show that suppression of the LH-secretory response to GnRH by
PRL and DA is accompanied by changes in mRNA synthesis, and
that the photoperiodic modulation of this inhibition operates
primarily at the level of LH release through alterations in PKC
and PLC. Furthermore, the suppressive effects of PRL and DA on
the secretion of FSH are photoperiodically regulated in a PT-
dependent manner.
dopamine, gonadotropin-releasing hormone (GnRH/GnRH
receptor), gonadotropins, LH, pituitary, prolactin/prolactin
receptor, seasonal reproduction, sheep
INTRODUCTION
In sheep, a short-day seasonal breeder, the annual patterns of
gonadotropin and prolactin (PRL) secretion are inversely
correlated, with highest concentrations of PRL detected during
the long days of summer, coinciding with the nonbreeding
season (NBS) [1–3]. Inhibitory actions of PRL on the
gonadotropic axis have been demonstrated, both at the level of
the hypothalamus to suppress gonadotropin-releasing hormone
(GnRH) secretion [4–6], and directly at the level of the pituitary
gland to suppress LH release [7–11]. Indeed, we have previously
shown that PRL suppresses the release of gonadotropins from
gonadotrophs in ovine pituitary cultures only when applied
concomitantly with bromocriptine (Br), a receptor-specific
agonist of dopamine (DA), the main hypothalamic inhibitory
regulator of PRL secretion [12]. Moreover, the suppressive
effects of PRL and DA are influenced by season, since they are
only apparent in cultures obtained during the summer (i.e.,
NBS). Coupled with morphological evidence for the specific
expression of both PRL receptors (PRLR) and dopamine-2-
receptors (DRD2) by gonadotrophs [11, 13, 14], these functional
findings provide clear evidence that a photoperiodically
regulated relationship exists between the gonadotropic and
lactotropic axes directly at the level of the ovine pituitary.
More recently, we have confirmed that application of both
PRL and Br also results in the suppression of the gonadotropin
response to GnRH in gonadotrophs derived from horse and
mice [11, 14, 15], representing a mechanism that is highly
conserved across species. Despite this, it is not known whether
the suppressive effects of PRL and Br on gonadotropin
secretion are confined to the level of hormone exocytosis or
are accompanied by changes in gene transcription. This is
particularly important in order to identify the level(s) at which
photoperiod prevents PRL and DA from suppressing gonad-
otropin secretion under short-day conditions in seasonal
breeders. Although the nature and origin of these photoperiodic
mechanisms remain elusive, they are presumably orchestrated
by melatonin, a neurohormone secreted by the pineal gland,
which encodes photoperiodic information. There is evidence
that the pars tuberalis (PT) of the pituitary gland plays an
important role in translating the signal encoded by melatonin
into a biological output that drives pars distalis (PD) function,
since it densely expresses melatonin receptors [16, 17], PRL
concentrations decrease following implantation of melatonin
directly into this tissue [18], and photoperiod continues to drive
PRL cycles in hypothalamo-pituitary-disconnected (HPD)
sheep [19, 20]. Therefore, the PT may be involved in the
photoperiodic regulation of the suppressive effects of PRL and
DA on the gonadotropin response to GnRH.
Using primary ovine pituitary cell cultures taken from ewes
during the BS and NBS, the aims of the present study were to:
1) investigate whether the suppression of the gonadotropin-
secretory responses to GnRH by PRL and Br are accompanied
by reciprocal changes in mRNA; 2) characterize the prelim-
inary signaling mechanisms underlying such effects; and 3)
explore the seasonal modulation of these effects through the
parallel preparation of cultures excluding cells from the PT.
MATERIALS AND METHODS
Tissue Collection
Ovine pituitary glands were obtained from ewes of mixed breeds during the
breeding season (BS) (December–January) and NBS (June–July). Animals
1Supported in part by the Biotechnology and Biological Sciences
Research Council, U.K., and the Royal College of Veterinary Surgeons,
U.K.
2Correspondence: Domingo J. Tortonese, Centre for Comparative and
Clinical Anatomy, University of Bristol, Southwell Street, Bristol BS2
8EJ, England, United Kingdom. E-mail: d.tortonese@bristol.ac.uk
3Current address: Department of Medicine, Section of Cell Biology,
Division of Diabetes Endocrinology and Metabolism, Imperial College
London, London SW7 2AZ, England, United Kingdom.
Received: 11 October 2011.
First decision: 10 November 2011.
Accepted: 18 January 2012.
? 2012 by the Society for the Study of Reproduction, Inc.
eISSN: 1529-7268 http://www.biolreprod.org
ISSN: 0006-3363
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were killed for commercial reasons at an abattoir (Baker’s, Nailsea, U.K.), and
the pituitaries dissected out immediately after death (n¼18/season). The stage
of the annual reproductive cycle was confirmed by careful examination of the
ovaries. During the BS, ewes were considered to be sexually active on the basis
of a recently formed corpus luteum, together with the presence of a large
follicle. In contrast, during the NBS, ewes were considered to be anestrus when
no corpora lutea, but a corpus albicans, was observed in the gonad. All
specimens were collected in accordance with the specified risk material
regulations of the United Kingdom (1997: dispatch of SRM for veterinary or
research purposes).
Ovine Pituitary Cell Culture
Ovine PD/PT primary pituitary cell cultures were produced following a
method described elsewhere [21, 22]. Briefly, for each experiment, pituitaries
from six animals were pooled, and 2-mm3blocks incubated in 12.5 ml
incomplete M199 containing 0.006 g collagenase and 0.006 g hyaluronidase
(Roche Diagnostics, Hertfordshire, U.K.) for 75 min at 378C in a shaking water
bath. Following incubation, the explants were immersed in Ca- and Mg-free
sterile PBS (Invitrogen, U.K.) containing 0.2 mM EDTA (Sigma-Aldrich,
U.K.), before dispersion in Ca- and Mg-free PBS. The supernatant was then
aspirated and an equal volume of complete M199 containing 10 U/ml
penicillin, 10 lg/ml streptomycin, 20 lg/ml gentamicin, 5 lg/ml insulin (all
Sigma-Aldrich), and 10% charcoal stripped lamb serum (Invitrogen) added.
The resulting suspension was then centrifuged (2500 rpm), the supernatant
removed, and the cells resuspended in complete M199 before plating at 400000
cells/well. The cells were incubated for 6 days at 378C and 5% CO2, and the
medium was replaced every other day. PD-only cultures were produced in an
identical manner, except for the removal of the PT using an excision with a
large margin to ensure that remnants of PT tissue were not present in PD
cultures and vice versa. Previous studies have demonstrated the validity of this
method for producing a reliable gonadotropin dose response to GnRH, and for
maintaining seasonal characteristics of ovine pituitary cells in vitro [12, 21].
Experimental Design and Strategy
To investigate the effect of PRL on the LH-synthetic and -secretory
responses to GnRH, wells from PD/PT cultures were assigned to one of the
following experimental groups: 1) medium (control, Con), 2) Br, 3) PRL, and
4) PRL þ Br. On Culture Day 6, treatments were applied according to their
designated experimental group and GnRH applied simultaneously for 90 min at
one of the following concentrations: 0, 1, or 10 nM. This incubation time has
previously been shown to lead to optimal LH release in an identical culture
system [12]. For each experimental group, five wells were assigned per dose of
GnRH. Br (a specific DRD2 agonist; Sigma-Aldrich) was administered at a
concentration of 10 nM; this dose of Br has been shown previously to suppress
endogenous PRL secretion from ovine primary pituitary cell cultures [12, 22].
To increase the concentration of PRL in the culture media, exogenous rat PRL,
which has previously been shown to react with ovine PRLRs [12, 22], was
administered at a concentration of 500 ng/ml (rat PRL-B-7, lot AFP-6452B;
National Institute of Diabetes and Digestive and Kidney Diseases [NIDDK],
Bethesda, MD). In the combined treatment (PRL þBr) group, rat PRL and the
DA agonist were used at the same doses as when applied alone. Due to tissue
constraints following removal of the PT, PD-only cultures were treated with: 1)
medium (control, Con), 2) Br, and 3) PRL þ Br. Following application of the
above treatments and GnRH to PD/PT and PD-only cultures, the medium was
removed and stored frozen for subsequent measurement of LH and FSH
concentrations by radioimmunoassay (RIA), and RNA was extracted from cells
(pooled from five wells) for quantification of gonadotropin subunits mRNA.
To assess the signaling pathways involved in mediating the suppressive
effects of PRL and Br on the LH response to GnRH during the NBS, the
following treatments were applied to ovine PD/PT NBS cultures: 1) control
(medium-alone), 2) control þ solvents (DMSO 1:1000 and tartaric acid
1:1000000), 3) PRL þ Br, 4) Bis-indolylmaleimide I (Bis-1; PKC inhibitor) þ
U73122 (PLC inhibitor), 5) Bis-1 þ Br, and 6) U73122 þ PRL. Treatments
were coapplied for 90 min with increasing doses of GnRH at 0, 1, and 10 nM.
For each treatment group there were four wells per dose of GnRH. Bis-1
(Calbiochem, U.K.) and U73122 (Sigma-Aldrich) were used at 10 lM and 2
lM, respectively. Similar concentrations have previously been shown to inhibit
specifically PKC and PLC activation in a range of models, including primary
pituitary cell and immortalized gonadotroph cultures [11, 23–25]. PRL and Br
were used at 500 ng/ml and 10 nM, respectively. Following treatment, media
were removed and stored at ?208C for quantification of LH concentrations by
RIA.
Polymerase Chain Reaction
Total RNA was extracted using the two-step guanidine thiocyanate-cyanate
method, and purity assessed by determining absorbance at wavelengths of 260
and 280 nm [26]. PCR for LHB subunit and FSHB subunit mRNA was
performed using a method previously described [11, 14]. Briefly, three sets of
primers were used: 1) primers specific to ovine LHB subunit (sense 50-
ATGGAGATGCTCCAGGGACTG-30and antisense 50-GCAGTCA
GTGCTGCTGAGGCG-30) [27] (PubMed accession no. X52488), 2) primers
specific to ovine FSHB subunit (sense 50-ATGAAGTCCGTCCAGTTCTGC-
30and antisense 50-CTTGCCGCAGTGACATTCAGT-30[28] (PubMed
accession no. X15493), and 3) primers specific to GAPDH (sense 50-
GAACGGGAAGCTCACTGGCAT-30, antisense 50-GTCCACCA
CCCTGTTGCTGTAG-30). Primers for the constitutively expressed gene,
GAPDH, were designed to span an intron to avoid amplification of genomic
DNA, and were used to confirm the integrity of the RNA and efficacy of the
PCR. Three controls were run in parallel: one in which water replaced the
RNA, one in which water replaced the RNA and reverse transcriptase omitted,
and one in which RNA was used but reverse transcriptase omitted. Due to
insufficient availability of RNA from PD-only cultures, PCR for FSHB subunit
mRNA was performed only in PD/PT cultures.
Hormone Assays
LH concentrations in ovine primary pituitary culture medium were
measured by a previously validated RIA [12, 29], with all samples being
assayed in duplicate. Iodinated ovine LH (LER-1056-C2) was used as tracer
with an ovine LH antibody (ASMN R 29; a gift from Prof. A.S. McNeilly,
Medical Research Council Human Reproductive Sciences Unit, Edinburgh,
U.K.) used at a working dilution of 1:120000. The LH standard (NIH-LHs 23;
NIDDK) was used over a range of 0.1–50 ng/ml. The limit of detection of the
assay (90% B/B0) was 0.29 ng/ml, and the intra- and interassay coefficients of
variation (CV) were 9.35% and 23.68%, respectively. FSH concentrations were
measured in duplicate by RIA [30] in a single assay. Ovine FSH (NIAMMD
oFSH-19) was used as tracer, with an ovine FSH antibody (NIDDK-NHI anti-
oFSH-1) employed at a working dilution of 1:12000. The standard (USDA-
oFSH-SIAFP-RP-2/AFP-4117A) was used over a range of 0.1–50 ng/ml. The
limit of detection of the assay was 0.14 ng/ml and the intra-assay CV was
13.12%.
Statistical Analyses
In both BS and NBS cultures, a total of four separate experimental groups
(three groups for PD-only cultures) were treated with GnRH. For each group,
five wells were assigned to each dose of GnRH, and the experiments were
repeated independently three times in each season. Each experiment was
undertaken using pituitaries pooled from 6 sheep, totaling 36 animals for both
seasons. No differences among replicates within season were observed. The
reported values in the present study represent the mean 6 SEM. The effects of
season, experimental treatment, and dose of GnRH on the LH and FSH
secretory and mRNA responses to GnRH from ovine primary pituitary cell
cultures were examined using multifactorial ANOVA. Because a statistically
significant season by treatment interaction was observed (Wilk lambda, P ,
0.001), two-way ANOVAs were then used to examine the effects of
experimental treatment and dose of GnRH on gonadotropin release and mRNA
expression within each season. To determine whether gonadotropin-synthetic
and -secretory responses to GnRH differed within and between treatment
groups with respect to controls, pairwise comparisons were made using the
Fisher post hoc test, and were considered significant at P , 0.05. For signaling
studies, an additional experiment was conducted in ovine primary cultures
using pituitaries pooled from six animals. The effect of treatment (i.e.,
inhibitors, solvents) and dose of GnRH on LH release were examined using
two-way ANOVA, followed by the Fisher post hoc test.
RESULTS
Effects of PRL and Br on the LH-Secretory Response to
GnRH in Ovine Pituitary PD/PT Primary Cultures During
the BS (Winter) and NBS (Summer)
During the BS, acute stimulation of PD/PT cultures with
increasing doses of GnRH resulted in a clear dose-dependent
LH release, with the maximal response observed at 10 nM
GnRH (6.4 6 0.3 vs. 31.6 6 0.8 ng/ml, for 0 and 1 nM GnRH,
respectively; P , 0.01) (Fig. 1A; BS). The application of PRL
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and Br, either alone or in combination, resulted in a significant
enhancement of the LH-secretory response to 1 nM GnRH (P
, 0.01) (Fig. 1, B–D; BS). Similarly, the secretagogue
increased LH release in PD/PT cultures derived from the
NBS (8.13 6 0.49 vs. 24.0 6 2.32 ng/ml, for 0 and 1 nM
GnRH, respectively; P , 0.01) (Fig. 1A; NBS), with maximal
stimulation observed in this case at 1 nM GnRH. By contrast,
whereas the application of PRL or Br alone was unable to alter
basal and stimulated LH release, the combined application of
PRL and Br completely abolished the LH response to all doses
of GnRH, as expected (P , 0.01) (Fig. 1D; NBS). Therefore,
the suppressive effects of PRL and Br on the LH-secretory
response to GnRH are regulated in a photoperiod-dependent
manner, being evident only in animals exposed to long days
(NBS).
Effects of PRL and Br on the LHB Subunit mRNA Responses
to GnRH in Ovine Pituitary PD/PT Primary Cultures During
the BS (Winter) and NBS (Summer)
During the BS, increasing doses of GnRH dose-dependently
increased LHB subunit mRNA expression in PD/PT cultures,
with the maximal response observed at 10 nM GnRH (P ,
0.01) (Fig. 2A; BS). The application of either PRL or Br
abolished the LHB subunit mRNA response to the secreta-
gogue (P , 0.01) (Fig. 2, B and C; BS), a suppression that was
not further enhanced when both compounds were applied
concomitantly (P . 0.05) (Fig. 2D; BS). During the NBS,
application of GnRH to PD/PT cultures resulted in an increase
in LHB subunit mRNA expression, with the maximal response
observed at 1 nM (P , 0.05) (Fig. 2A; NBS). This response
was blocked following the application of Br (P , 0.01) (Fig.
2B; NBS), but remained unaffected by the application of PRL
(P . 0.05) (Fig. 2C; NBS). The combined treatment of PRL
and Br, however, completely suppressed LHB subunit mRNA
(P , 0.01) (Fig. 2D; NBS).
Effects of PRL and Br on the LH Secretory and mRNA
Responses to GnRH in Ovine Pituitary PD-Only Primary
Cultures During the BS (Winter) and NBS (Summer)
During the BS, removal of the PT tended to enhance the LH
response to GnRH (6.38 6 0.3 vs. 33.36 6 4.89 ng/ml, for 0
and 1 nM GnRH, respectively; P , 0.01) (Fig. 3A; BS), an
effect that was not further enhanced following application of
Br, presumably because LH output was already maximal (Fig.
3B; BS). As observed for PD/PT cultures, the combined
application of PRL and Br did not suppress the LH response to
GnRH in PD-only cultures at this time of year. In contrast,
during the NBS, GnRH increased LH release in PD-only
cultures (6.93 6 0.18 vs. 29.2 6 4.07 ng/ml, for 0 and 1 nM
GnRH, respectively; P , 0.01) (Fig. 3A; NBS), and this effect
was completely suppressed following combined application of
PRL and Br (6.23 6 0.76 ng/ml, for 1 nM GnRH; P , 0.01)
(Fig. 3C; NBS), as previously seen in PD/PT cultures.
Likewise, removal of the PT had no affect on the increase in
LHB subunit mRNA expression that was observed in response
to GnRH irrespective of season (P , 0.01) (Fig. 4A; BS and
NBS). The application of Br alone was sufficient to block the
LHB subunit mRNA response to GnRH in cultures derived
during both the BS and NBS (P , 0.05) (Fig. 4B; BS and
NBS), and this effect persisted following the simultaneous
application of PRL (Fig. 4C; BS and NBS).
Signaling Pathways Implicated in the Suppression of the
LH-Secretory Response to GnRH by PRL and Br
Under control conditions, even moderate doses of GnRH
significantly increased LH release (9.1 6 0.2 vs. 27.4 6 3.6
ng/ml, 0 vs. 1 nM GnRH, respectively; P , 0.01) (Fig. 5A), an
effect that was not altered by application of solvents used to
dilute the chemical inhibitor compounds (Fig. 5B). As before,
combined application of PRL and Br abolished the LH
response to all doses of GnRH (P , 0.01) (Fig. 5C). These
inhibitory actions of PRL and Br could be mimicked using a
cocktail of Bis-1 and U73122, specific chemical inhibitors of
PKC and PLC, respectively (Fig. 5D). Confirming that
interactions between PLC and PKC were required for the
observed suppression by PRL and Br, the application of
U73122 þ PRL or Bis-1 þ Br similarly abolished the LH
response to GnRH (P , 0.01) (Fig. 5, E and F). Thus, the LH
response to GnRH is only suppressed when signaling via both
FIG. 1.
photoperiod-dependent manner. LH release from ovine pituitary PD/PT
primary cultures during the BS and NBS following treatment with:
medium (Con; A), Br (B), PRL (C), and PRL þ Br (D). The LH response to
GnRH administered at concentrations of 0, 1, and 10 nM is shown for
each experimental treatment group. Each bar represents the mean 6 SEM.
#P , 0.05 and
(ANOVA).
PRL and Br suppress the LH-secretory response to GnRH in a
##P , 0.01 versus same dose of GnRH in Con group
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the PKC and PLC pathways is concurrently downregulated by
PRL and DA (Fig. 6).
Effects of PRL and Br on the FSH-Secretory Response to
GnRH in Ovine Pituitary PD/PT Primary Cultures During
the BS (Winter) and NBS (Summer)
In PD/PT cultures derived during the BS, maximal FSH
release was detected in response to 1 nM GnRH (P , 0.05)
(Fig. 6A; BS). All treatments failed to affect the FSH response
to the secretagogue, except for combined application of PRL
and Br, which led to a slight but significant enhancement of the
FSH response to 1 nM GnRH (P , 0.05) (Fig. 6D; BS).
During the NBS, all doses of GnRH stimulated FSH secretion
(P , 0.01) (Fig. 6A; NBS), and application of either Br or PRL
alone selectively reduced the FSH response to high (10 nM)
concentrations of GnRH (P , 0.05) (Fig. 6, B and C; NBS).
Similar to the results for LH, the combined application of PRL
and Br completely abolished the FSH response to all doses of
GnRH (P , 0.01) (Fig. 6D; NBS).
Effects of PRL and Br on the FSHB Subunit mRNA Response
to GnRH in Ovine Pituitary PD/PT Primary Cultures During
the BS (Winter) and NBS (Summer)
During both seasons, GnRH failed to stimulate FSHB
subunit mRNA expression in PD/PT cultures, with high doses
of the secretagogue even reducing mRNA levels during the BS
(P , 0.05) (Fig. 7A; BS). In cultures derived during the BS, all
treatments failed to alter the FSHB subunit mRNA response to
GnRH, except for the combined application of PRL and Br,
which prevented the suppressive effects of 10 nM GnRH (P ,
0.01) (Fig. 7D; BS). Similarly, treatments had no effect in the
NBS, except Br alone, which suppressed the FSHB subunit
mRNA response to 1 nM GnRH below control values (P ,
0.01) (Fig. 7B; NBS).
Effects of PRL and Br on the FSH-Secretory Response to
GnRH in Ovine Pituitary PD-Only Primary Cultures During
the BS (Winter) and NBS (Summer)
In PD-only cultures obtained during the BS and NBS, FSH
release was maximal at 1 nM GnRH (P , 0.01) (Fig. 8A; BS).
Whereas application of Br alone was unable to affect the FSH
response to GnRH in cultures derived during the BS, the same
FIG. 2.
in a photoperiod-independent manner. LHB subunit mRNA expression in
ovine pituitary PD/PT primary cultures during the BS and NBS following
treatment with: medium (Con; A), Br (B), PRL (C), and PRL þ Br (D). The
LH response to GnRH administered at concentrations of 0, 1, and 10 nM
is shown for each experimental treatment group. Each bar represents the
mean 6 SEM.
(ANOVA).
PRL and Br suppress the LHB subunit mRNA response to GnRH
#P , 0.05 versus same dose of GnRH in Con group
FIG. 3.
modulation of the suppressive effects of PRL and Br on the LH response
to GnRH. LH release from ovine pituitary PD-only primary cultures during
the BS and NBS following treatment with: medium (Con; A), Br (B), and
PRL þBr (C). The LH response to GnRH administered at concentrations of
0, 1, and 10 nM is shown for each experimental treatment group. Each bar
represents the mean 6 SEM.##P , 0.01 versus same dose of GnRH in Con
group (ANOVA).
Removal of the PT does not modify the photoperiodic
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treatment enhanced the FSH response to all doses of
secretagogue when applied to cultures from the NBS (P ,
0.01) (Fig. 8B; BS). Intriguingly, excision of the PT appeared
to modulate the effects of PRL þ Br on FSH release, since, in
marked contrast to PD/PT cultures, suppression of the FSH-
secretory response to GnRH by the combined treatment was
present during both the BS and NBS in PD-only cultures (P ,
0.01) (Fig. 8C; BS and NBS).
DISCUSSION
The results of this study show that the previously reported
inhibition of the LH-secretory response to GnRH by the
concomitant actions of PRL and a DA agonist in ovine
pituitary cultures obtained during the NBS (summer) is
accompanied by reciprocal suppression of LHB subunit gene
expression. However, as the LHB subunit mRNA response to
GnRH was also suppressed by PRL and Br in cultures obtained
during the BS (winter), in the absence of treatment effects on
LH output, the current findings reveal that the photoperiodic
regulation of this inhibitory mechanism operates at the level of
hormone release. Moreover, such inhibition was shown to be
PT independent, because similar results were obtained for PD-
only cultures, devoid of PT-derived cell types. Interestingly,
removal of the PT allowed PRL and Br to suppress the FSH-
secretory response to GnRH during the BS, demonstrating that,
contrary to LH, blockade of this inhibition is photoperiodically
regulated in a PT-dependent manner for the FSH axis. The data
finally show that the inhibitory actions of PRL and DA on the
LH-secretory response to GnRH appear to require signaling
cross-talk at the levels of the PKC and PLC cascades,
providing a potential target for the photoperiodic mechanisms
that act to prevent these effects during the BS.
Interactions between the lactotropic and dopaminergic axes
that result in inhibition of pituitary gonadotropin secretion have
now been shown to operate in a number of mammalian species,
thus representing a highly conserved mechanism [11, 12, 14,
15]. Additionally, we have demonstrated that, in long- and
short-day breeders, PRL and Br suppress the LH response to
FIG. 4.
the LHB subunit mRNA response to GnRH. LHB subunit mRNA
expression in ovine pituitary PD-only primary cultures during the BS
and NBS following treatment with: medium (Con; A), Br (B), and PRL þBr
(C). The LH response to GnRH administered at concentrations of 0, 1, and
10 nM is shown for each experimental treatment group. Each bar
represents the mean 6 SEM.#P , 0.05 and##P , 0.01 versus same dose
of GnRH in Con group (ANOVA).
Removal of the PT does not alter the suppressive effects of Br on
FIG. 5.
for the suppression of the LH response to GnRH by PRL and Br. LH release
from ovine pituitary PD/PT primary cultures during the NBS following
treatment with: medium (Con; A), medium þ solvents used to dilute the
inhibitor cocktails (Con þ solvents; B), PRL þ Br (C), Bis-1 þ U73122
(specific inhibitors of PKC and PLC, respectively) (D), Bis-1 þ Br (E), and
U73122 þ PRL (F). The LH response to GnRH administered at
concentrations of 0, 1, and 10 nM is shown for each experimental
treatment group. Each bar represents the mean 6 SEM.##P , 0.01 versus
same dose of GnRH in Con group (ANOVA).
Signaling convergence at the levels of PKC and PLC are required
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GnRH only during the long days of summer [12, 15].
Therefore, the combined suppressive effects of PRL and DA
are dependent on season/photoperiod, and not stage of the
annual reproductive cycle. In the present study, we show that
this inhibition also operates at the level of gene expression, but,
in contrast to hormone release, suppression of LHB subunit
mRNA does not appear to be photoperiodically regulated.
Moreover, the application of Br alone was sufficient to
suppress the LHB subunit mRNA response to GnRH during
both the BS and NBS. Previous studies by our group using a
homogenous population of murine gonadotrophs (LbT2 cells)
have shown that PRL and Br in combination suppress both the
LH-secretory and mRNA responses to GnRH, and that,
whereas Br alone was able to suppress the LHB subunit
mRNA response to the secretagogue, it was unable to inhibit
LH output [11, 14]. Thus, the results of the present study
confirm these findings and, importantly, further demonstrate
that the combined suppressive effects of PRL and DA on the
LH response to GnRH are photoperiodically regulated directly
at the level of hormone release/exocytosis. The physiological
significance of such inhibition remains unclear, but it likely
plays different roles in long- and short-day breeders, even
though the circannual pattern of PRL is similar in both types of
species [1, 31, 32]. In sheep, the secretion of PRL is under
inhibitory regulation by hypothalamic DA neurons [32, 33],
noradrenaline [33], and pineal melatonin acting directly within
the pituitary [19, 20]. During the NBS, long day lengths are
encoded as short, nocturnal peaks of melatonin, leading to
disinhibition of PRL output through heterologous sensitization
of the melatonin receptor and its downstream pathways [34].
The resulting high concentrations of PRL detected during the
ovine NBS, in combination with DA, appears to ensure
complete inhibition of the gonadotropic axis at this stage of the
annual reproductive cycle. Although DA was reported to be
undetectable in the portal blood of ewes [35], the dopaminergic
FIG. 6.
photoperiod-dependent manner. FSH release from ovine pituitary PD/PT
primary cultures during the BS and NBS following treatment with:
medium (Con; A), Br (B), PRL (C), and PRL þ Br (D). The FSH response to
GnRH administered at concentrations of 0, 1, and 10 nM is shown for
each experimental treatment group. Each bar represents the mean 6 SEM.
#P , 0.05 and
(ANOVA).
PRL and Br suppress the FSH-secretory response to GnRH in a
##P , 0.01 versus same dose of GnRH in Con group
FIG. 7.
ovine pituitary PD/PT primary cultures during the breeding (winter) and
non-breeding (summer) seasons. FSHB subunit mRNA expression in ovine
pituitary PD/PT primary cultures during the BS and NBS following
treatment with: medium (Con; A), Br (B), PRL (C), and PRL þ Br (D). The
FSH response to GnRH administered at concentrations of 0, 1, and 10 nM
is shown for each experimental treatment group. Each bar represents the
mean 6 SEM.#P , 0.05 and##P , 0.01 versus same dose of GnRH in
Con group (ANOVA).
FSHB subunit mRNA expression is not stimulated by GnRH in
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tone is increased under long days in this species [36, 37], so
DA is likely to reach the pituitary via another route, such as the
pars nervosa [38], since treatment with sulpiride, a DRD2
antagonist, leads to PRL release in control, but not HPD, rams
[39].
As the suppressive effects of PRL and Br on the LH
response to GnRH were unaffected by removal of the PT, this
tissue seems unlikely to be required for transducing photope-
riodic information to the LH axis in the PD over the short term.
This was unexpected, given that the PT densely expresses
melatonin receptors and has been shown to be essential for the
photoperiodic control of PRL release from lactotrophs residing
within the PD [16, 17, 20, 40]. However, this does not rule out
the possibility that the PT is responsible for inducing long-term
alterations of PD function in vivo, which would persist for days
to weeks in vitro, especially given that seasonal responses are
chronic by nature. Indeed, it has previously been shown that,
following dispersion of pituitary glands obtained during the
NBS, high levels of PRL persist for at least 7 days [12],
providing evidence that the PT-based mechanisms that drive
PRL release in vivo evoke chronic changes in PD cell function,
which remain even ex vivo. However, to elucidate fully
whether this is the case, further in vivo studies would be
required to separate or ablate the PT, thus preventing
communication with the PD over a chronic time course.
Previous studies have demonstrated that GnRH increases
LH output via a PKC-MAPK3/1 signaling cascade, and PRL
suppresses the LH response to GnRH through inhibition of
these cascades directly at the level of the gonadotroph [11–13,
41, 42]. Conversely, the DRD2 is coupled to an inhibitory G
protein, which potently inhibits adenylyl cyclase, PLC, and
PKC activity [43]. In the present study, neither PRL nor Br was
able to suppress the LH-secretory response to GnRH when
applied alone; thus, simple inhibition of PKC or PLC activity is
unlikely to account for the suppression of the LH response to
GnRH observed following combined application of these
ligands. By employing a range of signaling pathway inhibitors,
we were able to show that the suppressive effects of PRL and
Br on the LH response to GnRH requires cross-talk between
their cognate receptors in the form of dual PKC and PLC
inhibition. This finding corroborates the results of a recent
study in our laboratory, which showed that the same ligands
abolished the LH-secretory response to GnRH in immortalized
murine gonadotrophs through signaling convergence at the
level of PKC to suppress ERK activation [11]. However,
although the PRLR is selectively expressed by the ovine
gonadotroph [13], in the present study, paracrine interactions
between DRD2-expressing lactotrophs and PRLR-expressing
gonadotrophs could not be ruled out, due to the nature of the
heterogeneous culture system used and the fact that the latter
cell type has been shown to be involved in the seasonal
regulation of PRL secretion [21]. Nevertheless, taken together
with the findings that murine and rat gonadotrophs express
both PRLR and DRD2, and that coapplication of PRL and Br
leads to suppression of both LHB subunit mRNA expression
and LH release in murine and ovine gonadotrophs [11, 12, 14],
it is reasonable to propose that activation of both these
receptors within the same cell underlies the inhibitory effects of
their ligands observed in the present study.
Of note is the observation that either PRL or Br alone was
able to suppress the LHB mRNA response to GnRH when
applied to PD/PT and PD-only cultures derived during the BS,
a finding that corroborates our results using immortalized cell
lines. The inhibitory effects of DA on PKC activity are well
described, and previous studies have shown that DA inhibits
LH mRNA expression in aT3-1 and LbT2 gonadotroph cell
lines through cAMP-dependent pathways [44, 45]. Therefore,
DA may abolish the LHB subunit mRNA response to GnRH by
suppressing PKC and cAMP, disrupting GnRHR coupling to
the MAPK pathway [41]. By contrast, we have previously
shown that PRL specifically inhibits the LHB subunit mRNA
response by suppressing phosphorylation/activation of ERK by
GnRH [11].
Regardless of whether or not the PT is involved, how does
season/photoperiod block the suppressive effects of PRL and
Br on LH release? It is unlikely that this inhibition results from
alterations in the expression of the PRLR or DRD2 by pituitary
gonadotrophs, because PRLR expression has been shown not
to change throughout the ovine annual reproductive cycle [13],
and PRL and Br were still able to suppress the LHB subunit
mRNA response to GnRH during both the BS and NBS.
Therefore, photoperiod may instead induce chronic alterations
in the function of signaling pathways, such as PKC, which
selectively sensitize the exocytotic machinery to calcium-
induced secretory granule release [46]. It is possible that
gonadal steroids are involved in this process, as pinealectomy
failed to prevent, but gonadectomy impaired, hyperprolactin-
emia-induced suppression of LH secretion [47, 48], and direct
effects of gonadal steroids on pituitary gonadotrophs are well
documented [49–52].
As for LH, the actions of PRL and Br on the FSH-secretory
response to GnRH were photoperiodically modulated, with
suppression of FSH output by the combined treatment only
FIG. 8.
suppressive effects of PRL and Br on the FSH response to GnRH. FSH
release from ovine pituitary PD-only primary cultures during the BS and
NBS following treatment with: medium (Con; A), Br (B), and PRL þBr (C).
The FSH response to GnRH administered at concentrations of 0, 1, and 10
nM is shown for each experimental treatment group. Each bar represents
the mean 6 SEM.#P , 0.05 and##P , 0.01 versus same dose of GnRH in
Con group (ANOVA).
Removal of the PT modifies the photoperiodic modulation of the
PROLACTIN AND DOPAMINE CONTROL OF GONADOTROPINS
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Page 8

observed during the NBS. However, and in marked contrast
with the LH axis, blockade of this inhibition was reliant on the
presence of the PT, since the same treatment completely
suppressed the FSH response to GnRH in PD-only cultures,
irrespective of season. Under control conditions GnRH was
unable to stimulate FSHB subunit mRNA expression in
cultures obtained during either the BS or NBS, perhaps as a
result of the time course of GnRH stimulation employed in
these studies, even though it did stimulate LHB subunit mRNA
in the same cultures. This highlights a differential response of
the two gonadotropins to the secretagogue, which precluded
the assessment of inhibitory effects of PRL and DA on FSH
gene expression. Nevertheless, the results from PD-only
cultures on FSH secretion imply that a PT-based mechanism
is active during the BS to prevent PRL and Br from
suppressing the FSH-secretory response to GnRH. While the
exact nature of this mechanism requires further characteriza-
tion, it is presumably photoperiodically regulated through the
actions of melatonin on its cognate receptor at the level of the
PT [16, 17, 20, 40]. These data raise the possibility that the PT
may contribute to the seasonal regulation of reproduction in
photoperiodic species by modulating the effects of PRL and
DA upon FSH release.
In summary, the results of the present study extend our
previous findings in various mammalian species, which show
that PRL is only able to suppress the LH response to GnRH
when applied in combination with a DRD2 agonist. Further-
more, photoperiod regulates these suppressive effects by acting
directly at the level of LH release/exocytosis. Given that PRL
and DA suppress LH release through receptor convergence at
the levels of PLC and PKC, factors released in response to the
ambient photoperiod must act to modify interactions between
these signaling pathways, by either preventing or promoting
the suppressive effects of PRL and DA during the BS and
NBS, respectively (see Fig. 9 for a schematic representation).
The results also demonstrate that PRL and DA similarly
suppress the FSH response to GnRH in a photoperiod-
dependent manner, but that, unlike LH, this action is regulated
by the PT. Taken together, these findings indicate that the
photoperiodic modulation of the intrapituitary control of
fertility by PRL and DA may represent an important and
conserved mechanism in the regulation of the seasonal
reproductive cycle.
ACKNOWLEDGMENT
We would like to thank Prof. A.S. McNeilly (Medical Research Council
Human Reproductive Sciences Unit, Edinburgh, U.K.) for generously
providing the LH antibody, and the NIDDKD’s National Hormone and
Peptide Program and Dr. A.F. Parlow (University of California, Los
Angeles) for providing RIA reagents and biologically active PRL.
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