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Department of Physiology, University of Adelaide (I.D.P., D.C.H., I.C.M.), Adelaide, South Australia 5005, Australia; and Department of Physiology, Colorado State University (R.V.A.), Fort Collins, Colorado 80523
Address all correspondence and requests for reprints to: Dr. Ian Phillips, Department of Physiology, Monash University, Clayton, Victoria 3168, Australia. E-mail: ian.phillips{at}med.monash.edu.au
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although circulating PRL concentrations are regulated by the fetal hypothalamus and the external photoperiod, the role of fetal PRL in the growth and development of fetal tissues during late gestation or in different photoperiod conditions remains poorly understood. It is clear from a range of studies that mRNA transcripts encoding long and short forms of PRL receptor (PRLR) are widely distributed in tissues derived from all three germ layers in the fetal rat (7) and mouse (8), and that immunoreactive PRLR is present in tissues derived from mesoderm and endoderm in the early human fetus (9). We have also recently demonstrated that the expression of mRNAs encoding the long (PRLR1) and short (PRLR2) forms of PRLR increase in the liver of the sheep fetus during the last 3 weeks of gestation (10). Anthony and colleagues (11) have shown that the sequence of the ovine PRLR2 complementary DNA (cDNA) is identical to that of ovine (o) PRLR1 cDNA until nucleotide 420, corresponding to E 261 in the oPRLR peptide-coding sequence (12). At this point, an insertion of 39 bases occurs between homology boxes 1 and 2 within the cytoplasmic domain of the receptor. The additional nucleotides in PRLR2 cDNA encode 11 amino acids followed by 2 stop codons (11, 12). Translation of PRLR2 mRNA is therefore predicted to encode a truncated or short form of PRLR. Although the long form of PRLR is able to transmit a full intracellular signal, the function of the short form of PRLR is not clear, although in vitro studies have shown that the ratio of long PRLR to short PRLR is important in intracellular signaling (13).
We also found that intrafetal administration of cortisol stimulated the expression of PRLR1 and PRLR2 mRNA in fetal sheep liver and therefore suggested that increases in hepatic PRLR expression before birth may be a result of the prepartum increase in fetal cortisol (10). PRLR abundance in adult rat liver has also been shown to be regulated by circulating PRL (14), but there is no information in other species, including the sheep, on the role of PRL in the control of PRLR gene expression. There is also no information concerning the role of the external photoperiod in the control of PRLR gene expression during development in seasonal species such as the sheep. In the present study we have therefore investigated the relative roles of the fetal hypothalamus, cortisol, and the external photoperiod in the regulation of PRLR expression in the liver of the sheep fetus. We used an animal model in which the fetal hypothalamus and pituitary are surgically disconnected [hypothalamo-pituitary-disconnection (HPD)] (4, 15). It has previously been demonstrated that surgical disconnection of the fetal pituitary from the hypothalamus does not alter the morphology, distribution, or proportion of lactotrophs in the fetal pituitary (15). Furthermore, the fetal PRL response to intrafetal administration of TRH and to alterations in the external photoperiod are maintained after HPD, providing evidence that the lactotrophs are fully functional after this procedure (16). It has also been demonstrated that HPD prevents the normal prepartum increase in fetal cortisol (16, 17). We compared the mRNA levels of PRLR1 and PRLR2 mRNA in the liver of the HPD fetal sheep (low PRL, low cortisol) with those in the HPD sheep fetus in which cortisol was infused (HPD + F; low PRL, high cortisol). Finally, we determined the effects of exposure of the ewe to different photoperiods on the expression of PRLR mRNA in the fetal liver. We measured hepatic PRLR1 and PRLR2 mRNA levels in late gestation fetuses from ewes maintained in either long (LL) or short (SL) photoperiods.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experimental protocols
HPD study. Ewes carrying intact (n = 7) or HPD fetuses
(n = 13) were housed under a 12-h light, 12-h dark cycle from the
time of entering the animal house at 95–110 days until postmortem. At
134, 135, or 136 d, cortisol (3.5 mg/4.8 ml sterile saline/24 h;
Solucortef, Upjohn, Kalamazoo, MI), was infused iv to five fetal sheep
in the HPD group for 5 days (HPD + cortisol group). Sterile heparinized
saline (5 ml/24 h) was administered iv to the remaining eight HPD fetal
sheep (HPD + saline group) and to seven intact fetal sheep (intact
group) for 5 days between 134/135 and 139/140 d.
Photoperiod study. Pregnant ewes (n = 12) were kept in a central holding facility between April and September (autumn and winter in the southern hemisphere) under a 12-h light, 12-h dark cycle (lights off at 1900 h) from at least 70 d, calculated from the time of mating, until surgery was performed between 106–120 d to implant fetal and maternal vascular catheters. Immediately after surgery, the ewes were housed in either a long photoperiod (LL group; 16 h of light, 8 h of darkness; n = 6) or a short photoperiod (SL group; 8 h of light, 16 h of darkness; n = 6) until 140–141 d. Two ewes in each lighting regimen carried twins.
Fetal blood sampling. Fetal arterial (2-ml) blood samples were collected into heparinized tubes at least three times per week between 0900–1200 h from 104–141 d. Plasma samples were prepared from fetal arterial blood by centrifugation at 1500 x g for 10 min at 4 C and stored at -20 C until required for measurement of PRL and cortisol. Fetal and maternal well-being were monitored by the collection of additional arterial blood (0.5 ml) from the fetus and ewe, and whole blood pH, PO2, PCO2, oxygen saturation, and hemoglobin content were measured using an ABL 550 acid base analyzer and an OSM2 hemoximeter (Radiometer, Copenhagen, Denmark).
Fetal liver collection. At 139–141 d, all ewes were killed by an iv overdose of sodium pentobarbitone. All fetal sheep (intact + saline group, n = 7; HPD + saline group, n = 8; HPD + F group, n = 5; LL group, n = 8; SL group, n = 8) were delivered by hysterotomy and weighed. Curved crown-rump lengths were measured in the LL and SL groups. Fetuses were killed by decapitation, and fetal livers (n = 33; intact + saline group, n = 6; HPD + saline group, n = 6; HPD + F group, n = 5; LL group, n = 8; SL group n = 8) were collected, weighed, snap-frozen in liquid nitrogen, and stored at -70 C.
PRL RIA. Plasma PRL concentrations were measured using rabbit anti-oPRL (anti-oPRL-2, batch AFP35810691, donated by the National Hormone and Pituitary Program, NIDDK, NIH, and obtained through Ogden Bioservices Corp., Rockville, MD) and a RIA previously described for use in fetal sheep plasma (3, 4). The sensitivity of the assay was 0.1 ng/tube, and the inter- and intraassay coefficients of variation were less than 20% and 10%, respectively.
Cortisol RIA. Total cortisol concentrations were measured in fetal sheep plasma by RIA using an Orion Diagnostica kit (Orion Diagnostica, Turku, Finland). Before assay, cortisol was extracted from the plasma using dichloromethane as previously described (18). The efficiency of recovery of [125I]cortisol from fetal plasma using this procedure was at least 90%. The sensitivity of the assay was 0.39 nmol/liter, and the cross-reactivity of the rabbit anti-cortisol antibody was less than 1% with cortisone and 17-hydroxyprogesterone and less than 0.01% with pregnenolone, aldosterone, progesterone, and estradiol. The inter- and intraassay coefficients of variation were always less than 10%.
Total RNA extraction from fetal liver. Total RNA was extracted from fetal liver samples (n = 29; intact + saline, n = 6; HPD + saline, n = 6; HPD + F, n = 5; LL, n = 7; SL, n = 5) by homogenization in 4 M guanidine hydrochloride and ultracentrifugation through a 5.7-M cesium chloride cushion. The purity and concentration of the nucleic acids were quantitated by spectrophotometric measurement as described previously (19).
Measurement of mRNA encoding long and short forms of PRLR:
synthesis of oPRLR-2 and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) riboprobes. Antisense complementary RNA (cRNA) oPRLR-2 and
oGAPDH riboprobes were transcribed in vitro as previously
described (11) by addition of the following reagents to a microfuge
tube: 4 µl 5 x transcription buffer [200 mM
Tris-HCl (pH 7.5), 30 mM MgCl2, 10
mM spermidine, and 50 mM NaCl], 2 µl 100
mM dithiothreitol, 0.8 µl RNA guard (Pharmacia Biotech, Sydney, Australia), 4 µl nucleotide
triphosphate mix (2.5 mM ATP, 2.5 mM GTP, and
2.5 mM CTP), 2.4 µl 100 µM UTP, 0.5 µg
(1.0 µl) linearized pCRII oPRLR-2 (11), 5 µl (50 µCi)
[
-32P]UTP (3000 Ci/mmol; Bresatec, Adelaide,
South Australia), and 1 µl (10 U) T7 RNA polymerase (Progen, Darra,
Queensland). The reaction was incubated at 37 C for 1 h, then DNA
template was removed by digestion with 1 U deoxyribonuclease 1
(Boehringer Mannheim, Sydney, Australia) for 20 min at 37
C. Newly synthesized cRNA was separated from unincorporated nucleotides
by column chromatography through Sephadex G-50 (Nick Column,
Pharmacia Biotech). The riboprobe complementary to a
portion of oGAPDH cDNA was synthesized by transcription from 50 ng
linearized pCRII oGAPDH in a reaction like that described above but in
which 2.5 µl (25 µCi) [-32P]UTP, 2.4 µl 500 µm
cold UTP, and 10 U (1 µl) T3 RNA polymerase (Progen) were
added to the reaction mix. The specific activities of newly synthesized
antisense PRLR-2 (660 bases) and oGAPDH (620 bases) riboprobes were
greater than 1 x 109 cpm/µg RNA and 1 x
108 cpm/µg RNA, respectively. The integrity and size of
the transcribed riboprobes were assessed by autoradiography after
electrophoresis of 30,000 cpm of each riboprobe on a 5% polyacrylamide
gel containing 8 M urea in 1 x TBE buffer (90
mM Tris, 89 mM boric acid, and 20
mM EDTA, pH 8). Radiolabeled cRNA transcripts of defined
length (Century Base Markers, Ambion, Inc., Austin, TX)
were also synthesized as described above and loaded into lanes (20,000
cpm/lane) on the gel. The gel was run in 1 x TBE buffer at 250 V
for 4 h, then dried on a vacuum gel drier and exposed to a
phosphorimager plate (BAS, Fuji Photo Film Co., Ltd.,
Tokyo, Japan) for 16 h. The image was visualized using a
phosphorimager machine (Fuji Photo Film Co., Ltd., BAS
1000) and MacBAS software (version 2.2).
Hybridization of total RNA and oPRLR and GAPDH riboprobes. Liver total RNA (10 µg) and two controls containing yeast transfer RNA (10 µg) were combined with antisense PRLR2 (3 x 105 cpm), GAPDH (1 x 105 cpm) riboprobes, and sterile water to a final volume of 20 µl. The RNA was precipitated by the addition of 2 µl 3 M sodium acetate (pH 5.2), 50 µl absolute ethanol, and incubation on dry ice for 15 min. The RNA was pelleted by centrifugation in a microfuge for 20 min at 4 C, and the supernatant was carefully removed. The RNA pellet was briefly air-dried, then resuspended in 20 µl hybridization buffer (80% formamide, 40 mM PIPES (pH 6.4), 400 mM NaCl, and 0.1 mM EDTA) and denatured by heating at 85 C for 4 min. Hybridization of the total RNA and riboprobes was carried out by overnight incubation at 45 C.
Ribonuclease (RNase) digestion. RNases (1 µg/ml RNase A and 12.5 U/ml RNase T1; Sigma Chemical Co., St. Louis, MO) in RNase buffer [300 mM NaCl, 10 mM Tris-HCl (pH 7.4), and 5 mM EDTA], was added (200 µl) to each hybridization, except for one control tube to which RNase buffer without RNases was added. Single stranded RNA was removed by incubation for 1 h at 30 C. The RNases were inactivated and RNA was precipitated by the addition of 300 µl solution Dx (Ambion, Inc.) and incubation at -20 C for 20 min. The RNA hybrids were pelleted by centrifugation in a microfuge for 30 min at 4 C, and all supernatant was carefully removed. The RNA pellet was briefly air-dried, and then resuspended in 8 µl gel loading buffer [80% formamide, 0.1% (wt/vol) xylene cyanol, 0.1% (wt/vol) bromophenol blue, and 2 mM EDTA]. The solution was heated at 85 C for 4 min, loaded onto a 5% polyacrylamide gel containing 8 M urea in 1 x TBE, and run as described above.
Detection and quantification of protected PRLR2 and GAPDH cRNA fragments. Autoradiographic images on the phosphorimage plate were visualized and quantified as described above. The antisense oPRLR2 cRNA probe (660 bases) was protected from RNase digestion by mRNA encoding a long and a short form of PRL receptor. The sizes of antisense PRLR2 cRNA fragments protected by mRNA encoding a long form of PRL receptor (PRLR1) were 440 and 73 bases. In this study, the relative amount of the 440-base probe fragment protected by PRLR-1 mRNA was quantified. mRNA encoding a short form of PRLR (PRLR2) protected 552 bases of PRLR2 antisense cRNA probe. GAPDH mRNA in fetal liver samples protected the antisense oGAPDH cRNA probe (650 bases) from RNase digestion. The signal output for PRLR2 and GAPDH riboprobes protected by PRLR1, PRLR2, and GAPDH mRNAs increased linearly with the input (10–30 µg) of liver total RNA, as previously described (10).
Statistical analyses
All data are presented as the mean ± SEM. The
mean plasma cortisol and PRL concentrations were calculated for each
fetus in the intact + saline, HPD + saline, and HPD + F groups during
the 5-day infusion period of either saline or cortisol. The mean plasma
PRL and cortisol concentrations for fetuses in the intact + saline, HPD
+ saline, and HPD + F groups were compared using one-way ANOVA. Where
the ANOVA indicated differences between groups, Fisher’s test ( =
0.05) was used post-hoc to identify which groups were
different.
The densitometric values for the 440- and 552-base fragments of PRLR2 cRNA probe in liver total RNA samples, protected by PRLR1 and PRLR2 mRNA from hydrolysis by RNases, were calculated as a ratio of the GAPDH cRNA signal in the same sample. Fetal body and liver weights, liver/body weight ratio, and PRLR1/GAPDH mRNA and PRLR2/GAPDH mRNA ratios in the intact + saline, HPD + saline, and HPD + cortisol groups were all compared using one-way ANOVA and Fisher’s post-hoc test.
A mean plasma PRL concentration was also calculated for each fetus within the LL and SL groups for the period between 110–141 d, and these values were compared using Student’s unpaired t test. Fetal body and liver weights, liver/body weight ratio, and PRLR1/GAPDH mRNA and PRLR2/GAPDH mRNA ratios in the LL and SL groups were compared using Student’s unpaired t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fetal cortisol and PRL concentrations
The mean plasma cortisol concentrations between 134–141 d were
significantly higher (P < 0.05) in the HPD + cortisol
group (97.4 ± 23.7 nmol/liter; n = 5 fetuses) than in the
intact + saline group (18.7 ± 3.8 nmol/liter; n = 5 fetuses)
or the HPD + saline group (1.5 ± 0.6 nmol/liter; n = 5
fetuses). The mean circulating PRL concentrations between 134–141 d in
the HPD + saline group (10.6 ± 4.3 ng/ml; n = 8 fetuses) and
the HPD + cortisol group (5.6 ± 2.0 ng/ml; n = 5 fetuses)
were lower (P < 0.05) than in the intact + saline
group (37.1 ± 6.8 ng/ml; n = 7 fetuses; Fig. 1
).
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). At 139–141 d, the relative amounts
of hepatic PRLR1/GAPDH mRNA and PRLR2/GAPDH mRNA (Fig. 3) were lower
(P < 0.05) in the HPD + saline group (10.6 ± 1.8
and 8.9 ± 1.8, respectively; n = 6) compared with those in
the intact + saline group (27.4 ± 6.1 and 17.7 ± 2.5
respectively; n = 6). Cortisol replacement after HPD increased the
relative amounts of PRLR1/GAPDH and PRLR2/GAPDH mRNA (23.4 ± 4.0
and 15.3 ± 3.0, respectively; n = 5) to levels that were not
different from those in the intact + saline group (Fig. 3). There were no differences among the
three groups in the abundance of hepatic GAPDH mRNA, measured in
arbitrary densitometric units (intact + saline, 792 ± 184; HPD +
saline, 811 ± 34; HPD + F, 985 ± 157).
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).
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). The mean ratios of hepatic PRLR1/GAPDH mRNA and PRLR2/GAPDH
mRNA in the LL group [4.58 ± 0.86 (n = 7) and 3.36 ±
0.36 (n = 7), respectively] were not different, however, compared
with those in the SL group [4.26 ± 0.79 (n = 4) and
3.02 ± 0.45 (n = 4), respectively; Fig. 4]. There was no difference in GAPDH
mRNA abundance (arbitrary densitometric units) in fetal liver between
the LL (170.1 ± 16.8) and SL groups (188.0 ± 26.6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously reported that there was an increase in the expression of the two forms of PRLR in the fetal sheep liver after 130 days gestation, coincident with the timing of the prepartum cortisol surge in the sheep (10). Furthermore, we found that intrafetal infusion of cortisol before 130 days resulted in 2.4- and 1.8-fold stimulation of hepatic PRLR1 and PRLR2 mRNA levels, respectively (10). Previous studies have shown a positive association between PRLR mRNA levels and lactogenic hormone binding in the liver of the adult rat (20).
The normal prepartum surge in fetal cortisol does not occur after the fetal hypothalamus and pituitary have been surgically disconnected earlier in gestation (16, 17), and in the present study, we found that hepatic expression of PRLR1 and PRLR2 mRNA was reduced by 67% and 50%, respectively, in HPD fetal sheep at 140 days gestation. Cortisol administration to the late gestation HPD fetus restored hepatic PRLR mRNA levels to levels comparable to those measured in intact fetal sheep at 140 days gestation. In contrast to cortisol, there was no relationship between circulating PRL concentrations and hepatic PRLR expression in the HPD or intact fetal sheep. It appears from our studies that the basal level of expression of PRLR in the fetal liver before 130 days gestation and after fetal HPD is relatively cortisol independent. There is, however, no increase in hepatic PRLR expression in the late gestation HPD fetus in the absence of cortisol replacement, and this suggests that the gestational increase is cortisol dependent. Although plasma cortisol concentrations were significantly higher in the HPD + cortisol group than in the intact group, the levels of hepatic PRLR1 and -2 mRNA were the same in each group. This may indicate that there is a specific range of circulating cortisol levels across which a hepatic PRLR response occurs. Alternatively, it has also been demonstrated that infusion of glucocorticoids stimulates the synthesis of corticosteroid-binding globulin in fetal sheep liver, and this could also act to limit the availability of cortisol within the fetal circulation (21). It is interesting that the expression of PRLR1 appeared marginally more sensitive than that of PRLR2 to the effects of HPD and subsequent cortisol replacement.
In the ovine fetus, the levels of mRNA encoding long and short forms of hepatic PRLR are comparable throughout late gestation (10), whereas in the liver of the pregnant rat, mRNA encoding a short form of PRLR predominates (22). These differences could be species dependent and may arise from the divergence in the structural organization of the ovine and rat PRLR genes (12). Alternatively, there may be differences in the requirements for PRL activity in fetal liver during development and in adult liver during pregnancy. The different amounts of mRNA encoding long and short forms of hepatic PRLR may influence the cellular response elicited by lactogenic hormones binding to the different forms of PRLR in the liver of the late gestation sheep fetus compared with those in the liver of the pregnant rat.
It is not yet known whether cortisol acts directly or indirectly to regulate hepatic PRLR mRNA levels in the fetal sheep. Although promoter regions of the rat PRLR gene have been identified, none contains a glucocorticoid response element (23), and there is no current information that defines the regulatory regions of the oPRLR gene. It is also possible that glucocorticoids may regulate PRLR transcription indirectly by modulating the transcriptional activity of gene(s)-encoding proteins, which, in turn, regulate PRLR gene transcription. Furthermore, although infusion of cortisol does not stimulate PRL synthesis and secretion, it is possible that cortisol acts in the intact fetus during early gestation or in the HPD fetus during late gestation to stimulate an increase in other hormones, such as thyroid or placental hormones (24), which, in turn, increase PRLR expression in the fetal liver. It has also recently been demonstrated, however, that expression of the GH receptor (GHR) and insulin-like growth factor I (IGF-I) mRNA increases between 130–140 days gestation in the fetal sheep liver, and that cortisol infusion before 130 days also stimulates hepatic GHR and IGF-I gene expression (25). PRLR and GHR are both members of the cytokine receptor superfamily and share common structural features, in that they possess a large extracellular domain, a single transmembrane region, and a cytoplasmic domain (26). It is possible, therefore, that regulation of PRLR, GHR, and IGF-I gene expression by cortisol plays an important role in the changes that occur in hepatic metabolism and somatotropic regulation either before birth or in response to intrauterine conditions such as chronic hypoxemia that result in an increase in circulating fetal cortisol concentrations (27).
There was a decrease in the size of the fetal liver relative to fetal body weight in the HPD group, and it was interesting that cortisol replacement increased the fetal liver/body weight ratio. Administration of PRL to rats causes hepatic hypertrophy (28); however, the relative roles of cortisol, PRL, and GH in hepatic growth and development before birth remain to be established.
Although PRL synthesis and secretion are regulated by the external photoperiod before and after birth in sheep (4, 5, 6), we found no evidence for a photoperiod effect on the expression of the mRNAs encoding either the long or the short form of the PRLR in fetal liver. Previous studies in the rat have found that several days of exposure to moderately elevated levels of PRL increase PRLR levels in the liver (26). It is possible that in sheep during late gestation, cortisol acts directly or indirectly to maximally stimulate PRLR levels, and therefore, PRLR expression in the fetal liver is relatively independent of the prevailing photoperiod and fetal PRL levels. The effects of the increased plasma concentrations of PRL in the sheep fetus during a summer gestation are unknown, although there are reports of seasonal differences in fetal growth profiles in sheep that are independent of differences in maternal nutrition (29).
In summary, we have demonstrated that expression of PRLR1 and PRLR2 mRNA levels in fetal liver are reduced after disconnection of the fetal hypothalamus and pituitary and are restored after cortisol replacement. In contrast to cortisol, there was no relationship in HPD or intact fetal sheep between circulating PRL concentrations and hepatic PRLR expression. In addition, we have also shown that PRLR1 and PRLR2 mRNA expression in fetal sheep liver are not regulated by changes in external photoperiod. These studies provide further insight into the potential role that the PRL axis may play in the transduction of signals about the external environment to the developing fetus as it prepares for the transition to extrauterine life.
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Acknowledgments |
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Footnotes |
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Received June 29, 1998.
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