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Department of Physiology, The University of Adelaide (D.C.H., I.C. McM.), Adelaide SA 5005; and Department of Physiology, Monash University (I.R.Y.), Clayton, Victoria 3168, Australia
Address all correspondence and requests for reprints to: I.C. McMillen, Department of Physiology, The University of Adelaide, Adelaide SA 5005, Australia. E-mail: cmcmillen{at}physiol.adelaide.edu.au
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ebling and co-workers (9) have also shown that the PRL response in the newborn lamb to an intermediate photoperiod of 12 h L, 12 h D depends on the photoperiodic history of the ewe. In lambs born to mothers that had been maintained in long photoperiods (16 h L) from 100 days gestation (term = 145 ± 3 days gestation), PRL concentrations were high at birth and then decreased rapidly within 14 days of exposure to 12 h L, 12 h D and remained low thereafter. In contrast, PRL concentrations in lambs born to mothers maintained on short photoperiods (8 h L), were low at birth and then gradually increased in the postnatal 12 h L, 12 h D photoperiod to exceed those in the group exposed to long photoperiods in utero (9). Similarly, studies in the juvenile male Siberian hamsters and other seasonal breeders have demonstrated that serum PRL concentrations and the reproductive response to an intermediate photoperiod are also dependent on the photoperiod experienced by the mother during late gestation (10, 11, 12, 13, 14). It has been inferred from such findings that in seasonal breeders, the fetus receives and responds to information about day length in utero and begins to develop a photoperiodic history before birth.
In the present study, we tested for the first time whether the lamb can construct a photoperiodic history in utero. We measured the fetal PRL response to an intermediate photoperiod (12 h L) in late gestation after exposure of the ewe to either a long (16 h L) or short (8 h L) photoperiod for 50 days in early pregnancy. We also investigated the relative roles of the fetal hypothalamus and pituitary in the measurement of photoperiodic history before birth. We previously have shown that the difference in fetal PRL concentrations in long and short photoperiods persists after surgical disconnection of the fetal hypothalamus and pituitary in late gestation (3). We have argued therefore that the effects of external photoperiod and maternal melatonin on fetal PRL secretion may be mediated at such extrahypothalamic sites as the melatonin-responsive cells of the pars tuberalis (3). In the present study, we investigated the effect of fetal hypothalamo-pituitary disconnection on the PRL response of the sheep fetus to a 12-h L photoperiod after prior exposure of the pregnant ewe to either a long or short photoperiod.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Surgery
Surgery was performed between 99–113 days gestation under
general anesthesia using halothane (0.5–4.0%) and
N2O:O2 (50:50 vol/vol) with aseptic techniques.
HPD was performed in the fetal sheep of 14 ewes as described in full
previously (3, 15). A midline incision was made in the fetal nose and
the nasal bone opened just left of the intranasal septum. The fetal
ethmoid and presphenoid bones were drilled to form a paramedian tunnel
beneath the anterior cranial fossa. The optic chiasm was located and
exposed to allow access to the median eminence. The neural tissue of
both internal and external laminae of the median eminence were removed
using gentle suction. A small piece of gelfoam soaked in thrombin
(Thrombostat: Parke-Davis, Caringbah, New South Wales, Australia) and
penicillin (Depomycin: Intervet, Lane Cove, New South Wales, Australia)
was introduced to separate the remaining hypothalamic tissue from the
pituitary. A sham procedure was carried out in 25 fetal sheep in which
either limited or no cranial dissection was performed, but in which
fetal vascular and amniotic catheters were inserted (sham group).
Catheters were inserted into a fetal and maternal carotid artery and
jugular vein, and into the amniotic cavity in all ewes. All catheters
were filled with heparinized saline, and the fetal catheters were
exteriorized via an incision in the ewes flank.
Blood sampling protocols after surgery
After surgery, all ewes were housed in metabolic cages and fed
alfalfa chaff (1 kg) once a day between 0900–1100 h, with water
available ad libitum. In 34 ewes, daily fetal (2.5 ml) and
maternal (5 ml) arterial blood samples were collected from the first
day after surgery until the end of the experimental period. In the
remaining 5 ewes, fetal and maternal arterial blood samples were
collected on every alternate day from day 6 or 7 after surgery. A total
of 920 fetal and 884 maternal blood samples were collected from the 39
pregnant ewes for PRL assay in this part of the study. Blood samples
were collected into heparinized tubes that were centrifuged for 10 min
at 1100 x g before separation and storage of plasma at
-20 C for assay. Fetal arterial blood samples (0.6 ml) were also
collected on each occasion for blood gas and pH analysis with an ABL
330 acid base analyzer and OSM 2 hemoximeter (both from Radiometer,
Copenhagen, Denmark).
TRH
TRH (50 µg; 15–25 µg/kg) was administered iv to fetal sheep
between 130–134 days gestation. Fetal arterial or venous blood (1.5
ml) samples were collected at -30, -5, +10, +20, +40, +60, and +120
min relative to the time of TRH administration. All blood samples were
collected into heparinized tubes that were centrifuged for 10 min at
1100 x g before separation and storage of plasma at
-20 C for assay.
Confirmation of HPD
Disconnection of the hypothalamus was confirmed in all HPD fetal
sheep as previously described (3) on the basis of the PRL responses to
intravascular chlorpromazine and on the basis of macroscopic
examination of the lesion site at postmortem. Chlorpromazine (CPZ, 12.5
mg; 4–6 mg/kg) was administered iv to fetal sheep between 132–140
days gestation. In the sham groups, fetal PRL concentrations were
50 ± 9 ng/ml (-5 min) and 116 ± 12 ng/ml (CPZ, +120 min)
(pre-LL) and 47 ± 13 ng/ml (-5 min) and 81 ± 16 ng/ml
(CPZ, +120 min) (pre-SL). In the HPD groups, however, fetal PRL
concentrations were 2.3 ± 0.9 ng/ml (-5 min) and 2.8 ± 1.1
ng/ml (CPZ, +120 min) in the pre-LL group and 6.8 ± 1.8 ng/ml
(-5 min) and 8.5 ± 2.3 ng/ml (CPZ, +120 min) in the pre-SL
group. Macroscopic examination of the lesion site postmortem confirmed
the completeness of the HPD procedure in all HPD fetal sheep.
PRL RIA
Plasma PRL was measured using rabbit antiovine PRL (Antiserum
batch number AFP 973269, generously donated by the National Hormone and
Pituitary Program, NIDDK, Baltimore, MD), and an assay that was
previously described and validated (3, 16). The sensitivity (defined as
the dose required to produce 10% displacement) of the assay was 0.1
ng/tube and the inter- and intraassay coefficients of variation were
<20% and <10%, respectively.
Postmortem
Thirty six ewes were killed between 136–144 days gestation
using an overdose of sodium pentobarbitone. Four ewes were killed
between 115–127 days gestation after sudden fetal death.
Statistical analyses
All results are expressed as the mean ± SEM.
Where the Cochrans and Bartlett-Box tests identified significant
heterogeneity of variance, maternal and fetal hormone concentrations
were logarithmically transformed before further statistical analysis.
The data were analyzed using the Statistical Package for Social
Scientists (SPSS Inc., Chicago, IL) and a VAX mainframe computer using
multifactorial ANOVA with repeated measures and photoperiodic history
(i.e. either pre-LL or pre-SL), treatment (i.e.
HPD or sham group), and the number of days spent in the 12-h L regimen
(in 5-day blocks) or gestational age (in 5-day blocks) as the specified
variables. PRL responses to TRH were also expressed as a percentage
change from baseline, (baseline values were taken as the mean of the
concentrations in the 30-min period before administration of TRH).
ANOVAs were used to determine whether there were significant changes in
PRL concentrations or in the percentage change from baseline in the TRH
experiments with photoperiodic history, treatment group, time (-30,
-5, +10, +20, +40, +60, and +120 min relative to time of drug
administration), and animal as the specified variables. Where the
multifactorial ANOVAs identified significant interactions between major
factors, the data were split on the basis of the interaction and
reanalyzed. When the ANOVAs indicated there were differences between
groups, the Duncan’s post hoc test was used to identify significant
differences between mean values. A probability of 5% (i.e.
P 
0.05) was taken to be significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Photoperiodic history: impact on fetal PRL response to a 12-h
photoperiod
Fetal PRL data were first analyzed to determine the effects of HPD
and photoperiodic history (i.e. pre-SL or LL) on fetal PRL
concentrations in a 12-h L regimen. The mean fetal PRL concentrations
were significantly lower (F = 20.1, P < 0.001) in
the HPD group (3.4 ± 1.0 ng/ml, n = 14 fetal sheep) than in
the sham fetal sheep (22.3 ± 4.0 ng/ml, n = 25 fetal sheep)
throughout the entire study period, i.e. between 105–144
days gestation. There was also a significant interaction (F =
11.5, P < 0.001) between the surgical treatment and
the effects of time spent in the 12-h L regimen, i.e. the
HPD and sham groups responded differently to the 12-h L regimen. The
data were split on the basis of surgical treatment group and
reanalyzed.
In the HPD group, there was a significant interaction (F = 6.3,
P < 0.001) between the effects of previous photoperiod
and the time spent in the 12-h L photoperiod on fetal PRL
concentrations. In HPD fetal sheep previously exposed to SL, fetal PRL
concentrations were significantly higher (P < 0.05)
after 20 days in the 12-h L regimen than previously (0–5 days,
3.2 ± 0.6 ng/ml; 21–25 days, 5.6 ± 1.4 ng/ml). In the HPD
fetal sheep previously exposed to LL, however, fetal PRL concentrations
significantly decreased (P < 0.05) during the first 5
days exposure to the 12-h L regimen (6.7 ± 2.9 ng/ml) and
remained low throughout the remaining study period (31–35 days,
1.7 ± 0.5 ng/ml) (Fig. 1a
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Photoperiodic history: impact on gestational age profile of fetal
PRL
The fetal PRL data from the HPD and sham fetal sheep were also
analyzed on the basis of gestational age. There was a significant
interaction (F = 11.9, P < 0.001) between the
effects of surgical treatment and gestational age. The data were split
on the basis of treatment group and reanalyzed.
In the HPD group there was a significant interaction (F = 6.9,
P < 0.001) between the effects of previous photoperiod
and the impact of gestational age on fetal PRL concentrations. In HPD
fetal sheep after exposure to SL, fetal PRL concentrations were
significantly higher (P < 0.05) after 130 days
gestation (6.7 ± 1.2 ng/ml) than earlier in gestation (< 110
days, 2.3 ± 0.4 ng/ml) (Fig. 1c). In HPD fetal sheep previously
exposed to LL, however, fetal PRL concentrations did not change
significantly with increasing gestational age (<110 days, 2.1 ±
0.5 ng/ml; 136–144 days, 2.5 ± 0.7 ng/ml) (Fig. 1c).
In the sham group, there was no effect of photoperiodic history on the
gestational age profile of fetal PRL, and PRL concentrations increased
significantly (F = 22.4, P < 0.001) in fetal
sheep previously exposed to either SL or LL. Fetal PRL concentrations
were significantly higher (P < 0.05) after 121 days
gestation in the 12-h L regimen in all sham fetal sheep (<110 days:
pre-SL 6.4 ± 0.3 ng/ml, pre-LL, 12.0 ± 3.3 ng/ml; 121–125
days: pre-SL 20.0 ± 3.9 ng/ml, pre-LL 25.9 ± 4.4 ng/ml)
(Fig. 1d).
Photoperiodic history and fetal PRL response to TRH
There was a significant fetal PRL response to TRH in both the HPD
(F = 20.9, P < 0.001) and sham (F = 31.3,
P < 0.001) groups (Fig. 2). There was
no difference, however, in the PRL response to TRH in fetal sheep
previously exposed to SL or LL in either the HPD or sham groups (Fig. 2). The maximum percentage changes in PRL occurred at +10 min after TRH
administration in the HPD (pre-SL, 421 ± 75%; pre-LL, 555
± 76%) and sham groups (pre-SL, 394 ± 68%; pre-LL, 369 ±
59%).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this and in previous studies we found that fetal PRL concentrations are lower in HPD than in intact fetal sheep, regardless of the prevailing photoperiod (3, 17). Surgical disconnection of the fetal pituitary from the hypothalamus does not alter the morphology, distribution, or proportion of the lactotrophs in the pars distalis (15). Furthermore, we showed that the proportional changes in fetal PRL from 10 min after TRH administration are similar in HPD and intact fetal sheep, which indicate that the lactotrophs in the surgically isolated pituitary are functional and responsive to external stimulation. It therefore appears that after HPD in fetal sheep, there is a loss of a predominantly stimulatory drive to PRL secretion. This loss may be a consequence of the lack of a hypothalamic PRL releasing factor after HPD. Alternatively there may be a loss of a placental signal that normally stimulates PRL secretion from the fetal pituitary and that depends on the presence of an intact hypothalamo-pituitary axis.
The findings of the present study indicate that the control of PRL secretion by the surgically isolated pituitary can be influenced by the photoperiodic history of the sheep fetus. Fetal PRL concentrations increased after 3–4 weeks exposure to an intermediate photoperiod (12 h L) in HPD sheep previously maintained in a short photoperiod (8 h L) regimen. Conversely, fetal PRL concentrations decreased within 6–10 days exposure to the intermediate photoperiod in HPD sheep previously maintained in a long photoperiod (16 h L) regimen. After 21–30 days exposure to the 12-h L regimen, PRL concentrations were around 3–5 ng/ml higher in the pre-SL than in the pre-LL HPD fetal sheep. If the HPD fetus simply responded to the absolute photoperiod, then the same pattern of PRL response to 12 h L might have been expected in the pre-SL and pre-LL groups. This did not appear to be the case, however, because the HPD fetal sheep maintained in pre-SL conditions responded to the 12 h L as a long day, whereas the pre-LL group responded to the 12 h L as a short day. In contrast, there was no measurable effect of photoperiodic history on the plasma PRL response to an intermediate photoperiod in the sham groups. In the sham animals, however, there was a consistent increase in the fetal plasma concentrations of PRL with increasing gestational age in both the pre-SL and pre-LL groups.
Although the effect of photoperiodic history on fetal PRL concentrations in the HPD groups was significant, it was relatively small, i.e. the greatest difference between the pre-SL and pre-LL groups was around 3–5 ng/ml after 21–30 days exposure to the intermediate photoperiod. It could be argued, therefore, that the effect of photoperiodic history on the PRL response to the intermediate photoperiod is present in the sham groups but is masked by the relatively greater stimulation due to factors acting on the fetal hypothalamus that are associated with increasing gestational age. It is important to note, however, that we found no evidence for a greater effect of photoperiodic history on the fetal PRL response to the intermediate photoperiod in the intact fetal sheep when compared with the HPD group. This implies that the effect of photoperiodic history on the fetal PRL response to an intermediate photoperiod is predominantly exerted through the fetal pituitary rather than the fetal hypothalamus.
In a recent study we demonstrated that plasma concentrations of PRL were higher in intact and HPD sheep fetuses exposed to long photoperiods (16 h L) compared with those exposed to short photoperiods (8 h L). These results are in agreement with those of Lincoln and Clarke (17) who demonstrated that there was photoperiodic regulation of PRL secretion in HPD rams in which the pituitary gland was functionally isolated from the brain. These authors also demonstrated that administration of melatonin to HPD rams under long days suppressed circulating PRL to values observed in HPD rams under short days (17). We concluded therefore, that in the sheep, differences in the prevailing external photoperiod and in the duration of the nocturnal increase in melatonin concentrations regulate PRL secretion by a pituitary dependent mechanism before and after birth. The present study also provides direct evidence that the surgically isolated pituitary has the capacity to generate a fetal PRL response that is influenced by the photoperiodic history of the ewe. It is likely that the reading of the photoperiodic history relies at least in part on the change in the duration of the nocturnal melatonin increase, which occurs on transition between the long or short days to the intermediate photoperiod. One possible extrahypothalamic site of action of melatonin is the pars tuberalis of the fetal pituitary, which remains intact after HPD. There is considerable indirect evidence that the pars tuberalis may be important in mediating seasonal reproductive responses to melatonin. In all mammalian groups, the pars tuberalis, unlike the pars distalis contains exceptionally high concentrations of iodo-melatonin binding sites (18). Most types of secretory cells in the ovine pars tuberalis are agranular, and it appears that these cells are melatonin responsive (19). One possibility is that the measurement of a photoperiodic history by these cells requires the stimulation or inhibition of the synthesis of a lactotrophic factor during exposure to long or short photoperiods, respectively, which is followed by a long-term decrease or increase in its secretion on transition to an intermediate photoperiod.
Ebling and co-workers (9) have previously demonstrated that prenatal photoperiod influences PRL secretion in the newborn lamb. In lambs born to ewes maintained on long days (16 h L) from 100 days gestation, circulating PRL concentrations were high for the first few days after birth, but fell rapidly to low levels within 14 days postnatally in 12 h L. Conversely, lambs born to ewes maintained on short days (8 h L) from 100 days gestation, had low PRL concentrations at birth but these gradually increased above 150 ng/ml by 30 days exposure to the 12-h L photoperiod. It is interesting that the difference between PRL concentrations in the pre-SL and pre-LL groups was greater (>100 ng/ml) in this latter study in the newborn lamb than that measured in our HPD or intact group of fetal lambs. There are a number of possible explanations for such a difference. The first may relate to the timing of exposure of the fetal lambs to the long and short photoperiods in these two studies. In the newborn lamb study, ewes were maintained in the long or short day regimens from around 100 days gestation until birth, whereas in our study, the ewes were maintained in similar regimens for around 50–57 days gestation. It has been established that plasma PRL concentrations are higher in 16-h L than in 8-h L regimens in intact fetal sheep after 100 days gestation (3, 20), but it is unknown whether this effect is present from as early as around 57 days gestation. Thus exposure to the different photoperiod regimens in early gestation does not necessarily imply that the fetal neuroendocrine system is able to monitor the photoperiodic environment from the time of exposure. It may also be that the greater apparent effect of photoperiodic history on neonatal PRL concentrations is simply related to a higher basal secretion of PRL in the newborn compared with the fetal lamb at the time of exposure to the intermediate photoperiod. Finally, direct exposure to light may amplify the effects of the intermediate photoperiod on the neuroendocrine system after birth compared with indirect exposure to the effects of the same photoperiod in utero.
Although this is the first study to investigate the neuroendocrine measurement of photoperiodic history directly in utero, there are a range of studies in other species, including the montane vole and Siberian hamster, that have shown that exposure to different prenatal photoperiods can influence the PRL and reproductive responses to intermediate day lengths postnatally (10, 11, 12, 13, 14). Studies in Siberian hamsters have shown that melatonin from the mothers’ pineal gland is involved in the prenatal transmission of the photoperiodic message to the pups, and that the message is related to the duration of the nocturnal melatonin increase in the mother (13). Interestingly, Shaw and Goldman (14) recently demonstrated that in the male Siberian hamster there is an influence of the prenatal photoperiod on the melatonin rhythm generating system in postnatal life, and they have suggested that this may contribute to the effect of prenatal photoperiodic history on postnatal secretion of FSH and PRL.
It is clear in the present study that there was a consistent effect of increasing gestational age on the fetal plasma concentrations of PRL in the sham group that was independent of the effects of prior photoperiodic history. We have previously demonstrated that there is an increase in PRL messenger RNA levels in the anterior pituitary of the sheep fetus between 130–141 days gestation (21). Further, the recent study of Houghton et al. (3) also demonstrated that fetal plasma concentrations of PRL increased progressively in late gestation with increasing exposure to either a long or a short photoperiod, which again provides evidence for a separate influence of gestational age on PRL secretion. Factors that may be important in the stimulation of PRL in late gestation include estrogens derived from the placenta. Estrogens stimulate PRL gene expression in pituitary lactotrophs in the adult sheep (22), and there is an increase in circulating oestrogens in the fetal sheep in late pregnancy (23). We have demonstrated that there was a significant effect of gestational length on maternal PRL concentrations that was also independent of the effects of prior photoperiod. The gestational increase in maternal PRL may also be related to the influence of placental estrogens in late pregnancy.
In summary, we demonstrated that there is an effect of photoperiodic history on the PRL response to an intermediate photoperiod in utero that does not require an intact and functional fetal hypothalamus. This effect, however, is either masked or suppressed by the stimulatory effect of factors associated with an increase in gestational age acting on the fetal hypothalamus. It appears from this study that although the fetal neuroendocrine system can construct a photoperiodic history, the prevailing photoperiod and gestational age exert relatively greater influences on fetal PRL secretion. Recent studies in the rat showed that PRL receptor messenger RNA is present in a range of key fetal organs, including the fetal liver, spleen, kidney, and muscle (24, 25). Definition of the factors that regulate the PRL axis in utero would enhance our understanding of the role that circulating PRL plays in fetal organ growth and metabolism in species that are responsive to changes in the external photoperiod.
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Acknowledgments |
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Footnotes |
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2 Recipient of a Commonwealth Postgraduate Research Award during
the conduct of this research.
Received September 6, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) previously exposed to SL photoperiod (a) and LL
photoperiod (b) and to sham fetal sheep (•) previously exposed to SL
photoperiod (c) and LL photoperiod (d).