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Department of Physiology (M.E.B., T.R.M., D.A.M.), College of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190; and Department of Physiology (T.J.M.), Cornell University College of Veterinary Medicine, Ithaca, New York 14850
Address all correspondence and requests for reprints to: Dr. Dean Myers, Department of Physiology, BMSB 653, University of Oklahoma Health Sciences Center, Post Office Box 26901, Oklahoma City, Oklahoma 73190. E-mail: dean-myers{at}uokhsc.edu
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Pituitary glands were collected from intact fetal sheep of four
gestational ages [100–107 days gestational age (dga), n = 8;
117–121 dga, n = 9; 126–130 dga, n = 9; 144–147 dga,
n = 8]. Lesions of the PVN (PVN Lx; n = 4) or sham lesions
(Sham; n = 5) were performed at 118–122 dga. Pituitary glands
from PVN Lx and Sham fetuses were collected at 139–142 dga (term
147 dga). POMC mRNA levels were determined by in situ
hybridization. POMC transcript levels were determined by both regional
analysis (20x magnification) and analysis of individual corticotropes
(400x magnification).
There was no difference among gestational age groups in superior anterior pituitary (AP) POMC mRNA levels determined by regional or cellular analysis. POMC mRNA levels were significantly greater in the inferior AP at 144–147 dga, compared with other gestational ages, using regional analysis (P = 0.003) or analysis of individual corticotropes (P < 0.01). POMC mRNA levels in the neurointermediate lobe in 126- to 130-dga fetuses were significantly greater than those in younger fetuses (P = 0.005) but not those in 144- to 147-dga fetuses.
There was no difference in POMC mRNA levels in the superior AP between PVN Lx and Sham, using regional analysis or analysis of individual corticotropes. In the inferior AP, there was a significant decrease in POMC mRNA levels in PVN Lx, compared with Sham, using both regional analysis (P < 0.01) and cellular analysis (P < 0.01). There was no difference in POMC mRNA levels in the neurointermediate lobe as the result of bilateral PVN Lx.
Our findings support that basal AP POMC mRNA levels are heterogenously
distributed in the ovine fetal AP, with POMC mRNA levels in the
inferior AP being significantly greater than in superior AP, by
144–147 dga. We further found that the higher POMC mRNA levels in the
inferior AP reflect significantly higher corticotrope POMC transcripts
and not simply a greater density of corticotropes in this AP region.
The increase in POMC mRNA levels at 144–147 dga in the inferior AP
seems unrelated to the onset of adrenocortical maturation (at
125–130 dga). Finally, we report that increase in corticotrope POMC
transcripts during late gestation in the inferior AP requires an intact
PVN.
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Introduction |
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125–130 days gestational
age (dga)] and continues through the final 3 weeks of gestation
(1, 2, 3, 4). During this time, adrenocortical steroidogenic enzyme
expression increases (5), as does adrenocortical sensitivity to
synthetic ACTH (6, 7). In adults, ACTH, produced and secreted from the
anterior pituitary (AP), is the major physiological regulator of
glucocorticoid production in the adrenal cortex (8). The onset of
adrenocortical maturation and length of ovine gestation can be modified
by manipulating fetal plasma ACTH levels. After 120 dga,
ACTH-treated fetal lambs deliver prematurely but prepared for life
ex utero by precociously elevated cortisol levels.
Conversely, gestation is prolonged in hypophysectomized fetuses, and
adrenocortical maturation is prevented. Both findings support ACTH as a
major regulator of fetal sheep adrenocortical activity. AP ACTH is subject to negative feedback control of secretion and biosynthesis of its precursor POMC by glucocorticoids (9, 10, 11). The fetal sheep paradoxically maintains peripheral plasma immunoreactive ACTH levels throughout late gestation, despite rising levels of plasma cortisol that would suppress adult ACTH secretion and synthesis (12). There is some disagreement as to whether fetal AP POMC messenger RNA (mRNA) levels are similarly maintained throughout the last third of gestation, in the face of increasing fetal plasma cortisol. Two reports from independent laboratories, using Northern analysis methodologies, showed increases in AP POMC mRNA within the final 10 days of gestation (13, 14). However, another laboratory twice reported a decrease in AP POMC mRNA in late gestation, between 135 dga and 141 dga, using similar methodologies (15, 16). Recently, Matthews et al. (17), using in situ hybridization (ISH) to analyze POMC mRNA levels in regions within the AP, described an increase in hybridization signal localized in the inferior region of the AP during the last week of gestation, suggesting a spatial heterogeneity in corticotrope distribution and/or in corticotrope POMC mRNA levels. However, no quantitative study of POMC mRNA abundance in individual corticotropes was attempted.
In fetal sheep, adrenocortical maturation and parturition are prevented by bilateral radiofrequency lesions of the fetal hypothalamic paraventricular nucleus (PVN) (18, 19). Dexamethasone implants adjacent to the PVN have a similar effect on adrenocortical maturation (5). Such disruptions of PVN function also abolish fetal plasma ACTH response to stressors (20, 21). In fetal sheep, as in adults, the parvocellular PVN expresses the ACTH secretagogues, arginine vasopressin (AVP) and CRH (13, 22, 23, 24). Controversy exists regarding the ability of CRH and/or AVP to regulate AP POMC mRNA levels in sheep (25, 26). The role of the fetal PVN in regulating AP POMC mRNA levels during gestation remains to be determined.
The purpose of the present study is to: 1) examine pituitary POMC mRNA levels during normal fetal development; and 2) determine whether bilateral destruction of the PVN alters basal levels of fetal pituitary POMC mRNA. Defining the ontogeny of POMC mRNA levels in individual corticotropes identifies populations of corticotropes that may play unique roles in adrenocortical maturation and the initiation of parturition. Analysis of PVN-Lx fetuses addresses whether basal POMC mRNA levels are independent of PVN function, or specific corticotrope populations are regulated by the PVN.
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Methods |
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Ontogeny of fetal pituitary POMC mRNA.
Ewes were
anesthetized by iv injection of ketamine (500–1000 mg). For each
fetus, the head and neck were delivered by cesarean section to allow
rapid exsanguination, then the entire fetus was removed from the
uterus. Pituitaries were collected within 10 min of exsanguination,
coated with Tissue Imbedding Medium (Triangle Biomedical Sciences,
Durham, NC), placed in isopentane, and immediately frozen by immersion
of the container into liquid nitrogen. Pituitaries were stored at -80
C until cryosectioned. Fetuses were obtained at 100–107 dga (n =
8), 117–121 dga (n = 9), 126–130 dga (n = 9), and 144–147
dga (n = 8).
Effect of lesion of the fetal PVN on pituitary POMC mRNA.
Radiofrequency lesions of the PVN were placed (n = 4), as
previously described (21), between 118 and 122 dga (term = 147
dga). In sham-lesion fetuses (n = 5), electrode tips were placed 5
mm above the vertical coordinate used in the lesion animals, without
activating the lesion generator. Maternal and fetal carotid and jugular
catheters were placed during surgery. A twin was removed from two ewes
(one lesioned, one sham). Blood gases were determined daily to monitor
fetal health. Beginning at 135–136 dga, blood samples (3 ml) were
drawn every 2 h for 60 h for use in another study. Red blood
cells were recovered under sterile conditions, chilled, and returned to
both fetus and ewe every 4–6 h. Fetal plasma ACTH and cortisol were
measured by specific RIA (Incstar, Stillwater, MN; DPC, Los Angeles,
CA) (27, 28).
At 139–142 dga, ewes were anesthetized with iv ketamine and maintained on halothane anesthesia. Fetuses were exsanguinated, and hypothalami and pituitary tissues were collected and prepared, as described above.
ISH
Tissues were cryosectioned at -18 C (20-µm sections,
hypothalamus; 25-µm sections, pituitary) and thaw-mounted on sialated
slides (Erie Scientific, Portsmouth, NH). Sections were stored at -80
C until ISH was performed. Lesion integrity was confirmed by
visualization of the PVN region, using prepro-AVP-neurophysin ISH of
the fetal hypothalamus, as previously described (13).
POMC mRNA levels were determined using ISH, as previously described (13). For each fetus, a series of sections spanning 4.5–5.0 mm of the long (antero-posterior) axis of the pituitary gland were analyzed. All pituitary sections in the ontogeny study were processed in a single ISH procedure (3 slides/fetus). Similarly, all pituitary sections from the PVN Lx study were processed in a single ISH procedure (nine to ten slides/fetus). To describe briefly, sections were fixed in paraformaldehyde, acetylated, dehydrated in an increasing series of ethanol solutions, delipidated, and air dried. Sections were prehybridized for 2 h at 37 C. Hybridization was then performed at 37 C for 18 h using an equimolar mixture of four oligonucleotide probes with similar melting temperatures (-315 to -276, -199 to -157, +13 to +54, and +324 to +363, where +1 = origin of translation) (29) at 106 cpm/slide (0.08–0.29 pmol each probe/slide in lesion and ontogeny experiments, respectively). Slides were then washed briefly in 1 x SSC (0.15 M NaCl; 0.015 M sodium citrate, pH7.2) at room temperature and twice in 1 x SSC at 55 C for 30 min. Slides were rinsed in 0.01% Triton X-100 in 1 x SSC at room temperature for 30 min and briefly immersed in 70% ethanol. After confirmation of hybridization by film autoradiography, slides were coated with nuclear emulsion (NTB 2, Eastman Kodak, Rochester, NY) and exposed at 4 C.
The ontogeny study compared neurointermediate lobe (NIL) and AP POMC mRNA hybridization between gestational groups. One emulsion-coated slide from each fetus was exposed for 9 h to optimize hybridization signal for quantification of POMC mRNA in the NIL. The remaining two emulsion-coated slides were exposed for 69 h to optimize hybridization signal for quantification of AP POMC mRNA. The latter slides included sections that were separated by at least 1.0 mm on the long axis of the gland. Emulsion-coated slides from sham and PVN Lx fetuses were exposed for 45 h to optimize quantification of AP POMC mRNA levels. Slides were developed and counterstained, as previously described (13).
Image analysis
Image analysis was performed using a 7100/66 PowerMac and public
domain NIH Image (Wayne Rasband, National Institutes of Health,
Bethesda, MD). Lightfield images were collected using an Olympus BX40
microscope equipped with a COHU high-performance CCD camera (RS170;
COHU Corp., San Diego, CA). Analysis of POMC mRNA hybridization signal
in the AP was performed on images collected, using 20x magnification
for regional analysis and 400x magnification for analysis of POMC mRNA
hybridization signal in individual corticotropes. Background
counterstaining (Toluidine Blue) was eliminated from images by the
following procedure: Each microscopic field was captured using
unfiltered illumination and using Wratten 4A (Kodak) filtered light. We
found that Wratten 4A filtering effectively eliminated the blue
counterstain from images without altering gray level of silver grains.
Features (pituitary regions or individual cells) to be analyzed were
outlined in images obtained with unfiltered light, then analyzed in
images obtained using Wratten 4A filtered light.
Quantity of POMC mRNA was determined using-25 µm sections of brain
paste standards containing known quantities of [
35S]
dATP. Standards were prepared, as previously described (13), and
simultaneously exposed to emulsion and subsequently developed with
experimental sections. Standards were imaged, as described above,
generating a gray-scale standard curve. Unknown grayscale values
obtained from imaged regions or individual cells were converted to POMC
mRNA (fmol/mg tissue) using the following formula: copies of
probe/µm3 = dpm/mg x (1.0 x 10-9
mg/µm3) x (1/specific activity of probe [dpm/mmol)) x
6.02 x 1023 copies/mol (13). Specific activity values
of oligonucleotide probes were determined individually and summed to
determine total specific probe activity.
Regional analysis of the AP was performed after the method of Matthews
et al. (Fig. 1
) (17).
Specifically, the maximum dorso-ventral dimension of the AP in each
coronal section was measured and the section divided into superior and
inferior halves by a straight line bisecting the dorso-ventral axis.
Hybridization of the total AP area was calculated by summation of
regional (inferior and superior) hybridization.
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and 3) at
400x magnification.
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Statistical analysis
Regional and cellular data were compared by one-way and two-way
ANOVA. Comparisons with P < 0.05 were analyzed by
Tukey-Kramer test. Those comparisons with 0.05 <P <
0.2 were analyzed by the Kolgorov-Smirnov one-sample test (two-sided),
and so noted. All results were expressed as mean ±
SEM.
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Results |
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) or cellular (Fig. 5B) analysis. There was a significant
increase in POMC mRNA levels in the inferior AP of 144- to 147-dga
fetuses, compared with all other groups using regional analysis
[P = 0.003; 0.234 ± 0.021 fmol/mg tissue
(144–147 dga) vs. 0.085 ± 0.012 (100–107 dga),
0.122 ± 0.014 (117–121 dga), and 0.108 ± 0.011 (126–130
dga)]. A similar increase in POMC mRNA was found in corticotropes in
the inferior region of the AP [P < 0.01; 0.490
± 0.079 fmol/mg tissue (144–147 dga) vs. 0.190 ±
0.039 (100–107 dga), 0.189 ± 0.029 (117–121 dga), and
0.236 ± 0.075 (126–130 dga)]. POMC mRNA levels in individual
corticotropes were significantly lower in the superior vs.
inferior regions in 144- to 147-dga fetuses (P <
0.01). This superior/inferior difference in corticotrope POMC mRNA
levels was not seen in other gestational age groups. No differences in
superior/inferior AP POMC mRNA hybridization were found in any
gestational age group using regional analysis. Total AP hybridization
was not significantly different among gestational age groups by either
regional (P = 0.07) or cellular (P =
0.07) analysis, when compared by ANOVA. When compared by the
Kolgorov-Smirnov test, total AP hybridization was significantly greater
in 144- to 147-dga fetuses, compared with 100- to 107-dga and 126- to
130-dga fetuses using cellular analysis [P = 0.025;
0.407 ± 0.016 fmol/mg tissue (144–147 dga) vs.
0.152 ± 0.007 (100–107 dga), 0.093 ± 0.021 (126–130
dga)]. When total AP POMC mRNA levels as determined by regional
analysis were compared by the Kolgorov-Smirnov test, the 144- to
147-dga group had greater hybridization than all other gestational age
groups [P = 0.05; 0.206 ± 0.057 fmol/mg tissue
(144–147 dga) vs. 0.071 ± 0.022 (100–107 dga),
0.0950 ± 0.026 (117–121 dga), 0.093 ± 0.021 (126–130
dga)].
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8.73 ± 1.01 fmol/mg tissue (126–130 dga) vs.
5.67 ± 0.33 (100–107 dga), 6.51 ± 0.91 (117–121 dga), and
7.62 ± 1.70 (144–147 dga)].
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There were no differences in fetal arterial PO2 (22.6 ± 3.1 vs. 21.9 ± 0.6 mmHg), PCO2 (42.3 ± 2.6 vs. 43.2 ± 3.7 mmHg), pH (7.376 ± 0.019 vs. 7.359 ± 0.013), or hemoglobin (13.1 ± 3.2 vs. 10.2 ± 0.7 g/dl) between lesioned and sham-lesioned fetuses. Similarly, plasma ACTH (22.4 ± 9.7 vs. 16.9 ± 5.2 pg/ml) and cortisol levels (13.9 ± 11.6 vs. 16.2 ± 7.8 ng/ml) were not different between lesioned and sham-lesioned fetuses and were similar to those previously observed in PVN Lx and sham-lesioned fetal sheep (18, 21).
There was no difference in POMC mRNA levels in the superior AP between
PVN Lx and sham-lesioned fetuses using regional analysis (Fig. 4A
; PVN
Lx: 0.61 ± 0.05, Sham: 0.49 ± 0.34 fmol/mg tissue) or
cellular analysis (Fig. 5A; PVN Lx: 1.78 ± 0.10, Sham: 1.88
± 0.16 fmol/mg tissue). In the inferior AP, there was a significant
difference between POMC mRNA levels in PVN Lx fetuses, compared with
those of sham-lesioned fetuses, using both regional analysis
(P < 0.01; PVN Lx: 0.76 ± 0.07, Sham: 3.17
± 0.66 fmol/mg tissue) and cellular analysis (P <
0.01; PVN Lx: 1.88 ± 0.20, Sham: 2.64 ± 0.36 fmol/mg
tissue). POMC mRNA levels were significantly different between
individual corticotropes in the superior vs. inferior AP in
sham-lesioned animals using cellular analysis (P <
0.01), and regional analysis (P < 0.05). There was no
difference between POMC mRNA levels in the inferior and superior AP in
PVN Lx animals using either regional or cellular analysis. Total AP
POMC mRNA was significantly decreased by PVN ablation when tested by
the Kolgorov-Smirnov one-sample test, using both regional analysis
(P = 0.01; PVN Lx: 0.69 ± 0.04, Sham: 2.50
± 0.77 fmol/mg) and cellular analysis (P = 0.05; PVN
Lx: 1.84 ± 0.28, Sham: 2.34 ± 0.50); no differences were
found using one-way ANOVA (P = 0.08 and 0.17,
respectively).
There was no difference in POMC mRNA levels in the NIL as the result of
bilateral PVN Lx (Fig. 6A).
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Discussion |
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The effect of corticotropin-releasing factors (such as CRH and AVP) on AP POMC mRNA levels in the sheep has been reported to vary with developmental stage. Corticotropes obtained from adult sheep respond to CRH with a slight increase in ACTH secretion, whereas exhibiting decreased POMC mRNA levels (25). Although AVP was a more potent ACTH secretagogue, it had no effect on POMC mRNA levels alone or administered in conjunction with CRH (25). Recent communications indicate that CRH (but not AVP) can increase POMC mRNA levels in dispersed cells obtained from 120- to 130-dga fetal sheep but not from near-term fetuses (26, 30). These in vitro studies indicate that CRH may regulate AP POMC mRNA levels in fetal sheep at certain gestational ages. The results from PVN Lx fetuses suggest that neuropeptide regulation of corticotrope POMC mRNA in vivo is different from its regulation in vitro. Our results indicate that neuropeptides originating from the fetal PVN sustain basal POMC mRNA levels in the AP in near-term fetuses. CRH and/or AVP may indirectly regulate basal POMC mRNA in the corticotrope in vivo by modulating functions of the glucocorticoid receptor. CRH has been shown to decrease glucocorticoid receptor expression in mouse corticotrope tumor cells (31). In addition, there may be factors absent in the in vitro model critical for regulation of corticotrope POMC mRNA, such as neuroendocrine modulators or corticotrope contact with neighboring cells (32). Such factors could promote POMC mRNA synthesis or stability or reduce the sensitivity of corticotropes to negative feedback from rising glucocorticoid levels.
The difference between inferior and superior AP POMC mRNA levels found in the sham-lesioned fetuses (in contrast to the PVN Lx fetuses) suggests that the spatial heterogeneity of POMC mRNA distribution in the AP is dependent upon an intact PVN. The PVN is known to regulate AP hormone expression by humoral factors secreted into the pituitary portal system at the median eminence. The concentration of POMC mRNA levels in the richly vascularized inferior AP may be a result of relatively high blood flow, compared with the superior AP, with consequently more exposure to hypothalamic factors, such as CRF and AVP. The fetal sheep AP also contains nerve fibers from the hypothalamus, including a network of AVP-immunoreactive nerve fibers concentrated in the medial portion of the gland closely associated with ACTH-staining cells (33). Thus PVN ablation may decrease humoral factors or innervation that support spatial heterogeneity in fetal AP POMC mRNA levels.
Our findings that the PVN is required for normal basal POMC mRNA levels in the AP of 139- to 142-dga fetal sheep contrasts with results from similar experiments in the adult rat in which AP POMC mRNA levels were unchanged by PVN ablation (34). The difference suggests that in the near-term fetal sheep (unlike the adult rat), maintenance of basal AP POMC mRNA levels is dependent on input from the PVN. Alternatively, basal near-term fetal AP POMC mRNA levels may be the sum of constitutive expression plus an elevation in POMC mRNA caused by chronic intermittent hypoxia and mechanical stress from increasing prepartum uterine motility. Short-term (5 h) hypoxia has been shown to increase ovine fetal AP POMC mRNA in the inferior region at 135 dga (35). Myometrial contractile activity can increase fetal plasma ACTH, an effect that is blocked by maintaining fetal normoxemia (36). Whether basal AP POMC mRNA levels in the term fetal sheep are regulated differently than in the adult or are the sum of constitutive and stimulated components, an intact PVN is required.
Ovine fetal NIL POMC mRNA levels were 2.6–13x higher than in the AP, which agrees with the work of previous investigators (17). The increase seen in NIL POMC mRNA in the 126- to 130-dga gestational age group conflicts with the previous study, which may be caused by differences in gestational ages analyzed. Alternatively, shortened exposure of emulsion to optimize NIL visualization allowed us to use a linear standard curve and avoid silver grain saturation over the NIL.
The contribution of the NIL to fetal adrenal development and maturation
has been under investigation since ACTH immunoreactivity was first
detected in the NIL (37). A role for the hypothalamus as a possible
positive regulator of NIL POMC expression has been suggested recently.
The presence of CRH-immunostaining in the rat NIL in vivo
has been noted by several investigators (38, 39). CRH has been shown to
increase POMC mRNA levels (40) and melanocyte stimulating hormone
(MSH) secretion in rat melanocytes in vitro (41).
Recently, CRH has been reported to modulate ACTH secretion from
dispersed fetal sheep NIL/neuro-hypophysis cultures (42). The increase
in NIL POMC mRNA in 128- to 130-dga fetuses of our ontogeny study
occurs during a period of increased PVN CRH mRNA levels (13). Thus NIL
POMC mRNA levels may be regulated, in part, by hypothalamic CRH.
Because fetuses in the PVN Lx study were collected at 139–142 dga, it
is not known whether NIL POMC mRNA levels in lesioned fetuses differed
from sham-lesioned animals at 128–130 dga. Previous studies in
hypothalamo-pituitary disconnected fetal sheep have shown that the
hypothalamus has a strong inhibitory effect on NIL size and POMC mRNA
levels (43). The PVN apparently is not the major inhibitor of NIL size
and POMC mRNA levels in the fetal sheep at 139–142 dga, because these
NIL parameters were not different between PVN Lx and sham-lesioned
animals.
In summary, fetal AP POMC mRNA levels increase between 130 and 144 dga; this increase fails to occur in fetuses subjected to PVN Lx at 118–122 dga. Thus an increase in AP POMC mRNA is not associated with the onset of adrenocortical maturation. It occurs in the last days of gestation and requires an intact fetal hypothalamic PVN.
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Footnotes |
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Received February 12, 1997.
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This article has been cited by other articles:
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  M. Keller-Wood, M. J. Powers, J. A. Gersting, N. Ali, and C. E. Wood Genomic analysis of neuroendocrine development of fetal brain-pituitary-adrenal axis in late gestation Physiol Genomics, February 23, 2006; 24(3): 218 - 224. [Abstract] [Full Text] [PDF]
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D. A. Myers, M. E. Bell, T. J. McDonald, and T. R. Myers Corticotropin-Releasing Factor Receptor Expression in the Pituitary of Fetal Sheep after Lesion of the Hypothalamic Paraventricular Nucleus Endocrinology, September 1, 1999; 140(9): 4292 - 4299. [Abstract] [Full Text]
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M. E. Bell, T. R. Myers, and D. A. Myers Expression of Proopiomelanocortin and Prohormone Convertase-1 and -2 in the Late Gestation Fetal Sheep Pituitary Endocrinology, December 1, 1998; 139(12): 5135 - 5143. [Abstract] [Full Text] [PDF]
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