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DEVELOPMENTAL PHYSIOLOGY AND PREGNANCY
1Department of Obstetrics and Gynecology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma; and 2Center for Perinatal Biology, Loma Linda University School of Medicine, Loma Linda, California
Submitted 12 October 2004 ; accepted in final form 19 December 2004
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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ACTH; adrenocorticotropin; anterior pituitary; sheep
Several methods have been used to induce experimental hypoxia in the sheep fetus, with the duration ranging from acute (minutes to hours) to prolonged (several hours to several days) to a few weeks (21). Our laboratory has developed a model of long-term hypoxia (LTH) in which pregnant ewes are maintained at high altitude (3,820 m) from day 30 of gestation until near term. The LTH fetuses exhibit basal plasma concentrations of IR-ACTH and cortisol similar to those of normoxic controls (1, 14). However, in response to acute secondary stressors, such as hypotension (1) or umbilical cord occlusion (14), fetal plasma cortisol is elevated in LTH compared with normoxic control fetuses, whereas plasma IR-ACTH levels attain similar concentrations in response to the secondary stressor. Furthermore, LTH fetal sheep demonstrate a decreased cortisol response to exogenous ACTH (ACTH1–24) compared with control fetuses (13).
In addition to ACTH, the ACTH precursor proopiomelanocortin (POMC) and POMC-derived processing intermediates containing the ACTH sequence are also found in fetal plasma. ACTH precursors are observed at 
10-fold higher concentrations in the fetal circulation compared with ACTH (8, 25, 34) and, because they contain the ACTH moiety, display varying degrees of cross-reactivity with various ACTH antisera used in RIAs, thus making accurate assessments of ACTH1–39 difficult using classical RIA technology. Both POMC and 22-kDa pro-ACTH have been shown to attenuate ACTH-induced glucocorticoid production by ovine fetal adrenocortical cells (30); 22-kDa pro-ACTH is the major processing intermediate for ACTH and consists of the NH2 terminus of POMC through the carboxyl residue of ACTH. A decrease in the ACTH precursor-to-ACTH ratio during late gestation is consistent with reports of an enhanced biological activity of IR-ACTH in fetal plasma as term gestation approaches (6, 9, 33, 34). Furthermore, acute stress has been reported to significantly enhance the biological activity of fetal plasma IR-ACTH (6, 10, 17, 29, 34). These studies indicate that anterior pituitary processing of POMC to ACTH increases with advancing gestational age and acutely under hypothalamic neuropeptide regulation.
From our group's previous findings that LTH fetal sheep exhibit an enhanced cortisol response to secondary stressors and that fetal plasma IR-ACTH concentrations are similar (1, 14), we hypothesized that anterior pituitary corticotrope processing of POMC to ACTH would be enhanced in LTH fetal sheep, and this would be reflected by a reduction in anterior ACTH precursors and ACTH with a significant increase in fetal plasma ACTH relative to ACTH precursors. Because there is evidence that suggests neuroendocrine regulation of POMC processing and the bioactivity of fetal plasma ACTH and the ability of a CRF type 1 receptor (CRF1-R) antagonist to suppress fetal plasma ACTH and delay parturition (11, 16), we also examined hypothalamic CRF expression and expression of the CRF1-R in the anterior pituitary of LTH fetal sheep.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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All studies were conducted with approval of the Loma Linda University School of Medicine Institutional Animal Care and Use Committee. Fetal sheep were obtained from pregnant ewes maintained at sea level (300 m, maternal arterial PO2 = 102 ± 2 Torr) or at high altitude (Barcroft Laboratory, White Mountain Research Station, Bishop, CA; altitude 3,820 m, maternal arterial PO2 = 59.1 ± 5.4 Torr) for 110 days beginning on day 30 of gestation (term =146 days). Animals from the latter group were then transported to Loma Linda Medical Center, at which time a nonocclusive maternal tracheal catheter was placed. Reduced PO2 was maintained at a level comparable to that observed at altitude by nitrogen infusion as previously described (1, 13, 14). On days 139–141 of gestation, the ewes were sedated with pentobarbital sodium, intubated, and maintained under general anesthesia with 1.5–2% halothane in oxygen, while the fetuses were delivered through a midline laparoptomy. Pituitaries were then obtained, and anterior pituitaries were quickly separated from posterior pituitary/intermediate lobes, frozen in liquid nitrogen, and stored at –80°C until analysis. Hypothalami were also collected and snap frozen. We dissected the hypothalami using the anterior aspect of the optic chiasm as the forward boundary, posterior 1 cm to the mamillary body and vertically 1 cm. Basal plasma samples for determination of ACTH precursor and ACTH concentrations were collected from control and LTH chronically catheterized fetuses from a previously described study (14).
Western Blot Analysis of ACTH Precursors
Tissue. Anterior pituitaries (n = 5 control, n = 5 LTH) were homogenized (4°C; 1 ml; 0.1 M acetic acid, 100 mM sodium chloride, pH 5.0, containing 1 mM pepstatin, 0.4 mM pefablock, and 1 µg/ml leupeptin) and centrifuged at 12,000 g for 2 min, and the supernatant was collected. Protein concentrations were determined by the Bio-Rad method.
Immunoprecipitation of ACTH and precursors. Immunoprecipitation was performed with 100 µg of protein/anterior pituitary sample. The pH of each homogenate (100 µg of protein) was adjusted to 7.2 by using 1 M Tris base, and samples were brought to 250 µl. Sodium chloride and Tris concentrations were adjusted with 1-M stocks to ensure that each 250-µl sample was 20 mM Tris (pH 7.2) and 100 mM NaCl. BSA (10% stock solution) was added to each sample to a final concentration of 0.1% (vol/vol), and Triton X-100 was added to a final concentration of 0.1%. One microgram of each monoclonal antibody against ACTH (monoclonal anti-ACTH1–24 and anti- ACTH16–39; Biodesign, Kennebunk, ME) was added to the anterior pituitary extracts, and the samples were then incubated overnight at 4°C with mixing. Immunoprecipitation was accomplished by the addition of 1 µl of horse anti-mouse serum (Vector Laboratories, Burlington, VT) followed by incubation for 30 min at room temperature with mixing. The volume was adjusted to 1 ml with 20 mM Tris·HCl and 100 mM sodium chloride, and the immune complex was subsequently precipitated by centrifugation at 12,000 g at 4°C for 15 min. The supernatant was removed by aspiration, the pellet was washed once with PBS and centrifuged again at 12,000 g for 5 min (4°C), and the immune complex subsequently was solubilized with 30 µl of SDS-PAGE loading buffer.
Western blot analysis. ACTH and ACTH-containing precursors were electrophoresed by SDS-PAGE (10–20%; Invitrogen, Carlsbad, CA) without reducing agent, and the proteins were subsequently electrophoretically transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA) and subjected to Western blot analysis. The membrane was blocked for 1 h with 10 mM Tris·HCl, pH 7.2, and 100 mM saline containing 0.1% Tween 20 (TTBS) and 10% nonfat dry milk (NFDM). The membrane was then washed twice in TTBS and incubated with the primary antibody (horseradish peroxidase-labeled anti-ACTH1–24 IgG; Sangui Biotech, Santa Anna, CA) prepared in TTBS, 5% NFDM overnight at 4°C. A chemiluminescent detection system (Pierce) was used, and blots were exposed to film (Hypermax) for varying lengths of time. Controls were 1) preabsorption with excess ACTH1–24; 2) use of ovine recombinant POMC, 22-kDa pro-ACTH, and synthetic ACTH1–39; and 3) a control tissue as source of protein for immunoprecipitation (liver). The immunoprecipitation and Western blot analysis were performed three times for all samples, and sample values were expressed as integrated densiometric units.
Western blot analysis of CRF1-Rs. Extracts (50 µg) of anterior pituitary prepared as described above were diluted 2:1 with loading buffer, boiled for 5 min, and then subjected to SDS-PAGE (10–20%; Invitrogen) under reducing conditions; the proteins were subsequently electrophoretically transferred to nitrocellulose membranes (Bio-Rad) and subjected to Western blot analysis. The membrane was blocked for 1 h with TTBS and 10% NFDM. The membrane was then washed twice in TTBS and then incubated with the primary antibody (1:1,000; rabbit anti-human CRF1-R; generously donated by Elizabeth Linton, Oxford University, UK) prepared in TTBS, 5% NFDM overnight at 4°C. A chemiluminescent detection system (Pierce) was used, and blots were exposed to film (Hypermax) for varying lengths of time. Controls were 1) use of ovine recombinant CRF1-R (23) and 2) a control tissue (liver).
Immunoassay of Plasma ACTH1–39 and ACTH Precursors
Fetal plasma ACTH1–39 was measured using a two-site immunoradiometric assay (IRMA; DiaSorin, Stillwater, MN) with a sensitivity of 1 pM. Parallelism of the assay was determined in our laboratory using ovine fetal plasma and fetal plasma to which known amounts of ACTH1–39 had been added. Intra-assay coefficient of variation for this study was 5.2%; all samples for this study were analyzed in the same assay. As described by the manufacturer, this IRMA shows no cross-reactivity against 
-, 
-, or 
-MSH or -endorphin. During the validation of the assay, we determined that the ACTH IRMA exhibited <0.1% cross-reactivity against POMC precursors (recombinant ovine POMC and 22-kDa pro-ACTH and the human ACTH precursor standard obtained from the assay described below to measure ACTH precursors).
We measured fetal plasma ACTH precursors using a specific two-site ELISA (OCTEIA POMC, IDS Limited, American Laboratory Products, Windam, NH) with a sensitivity of 1.0 pM. As described by the manufacturer, this ELISA exhibits <0.1% cross-reactivity against - or -MSH, <2.2% against -MSH, and <0.1% against ACTH1–39 or ACTH1–24. During the validation of the assay, we confirmed the cross-reactivity against - and -MSH and ovine ACTH1–39 and ACTH1–24. Intra-assay coefficient of variation for this study was 4.5%. Parallelism and recovery for the assay were determined in our laboratory for ovine fetal plasma spiked with known amounts of POMC standard from the ELISA kit and recombinant ovine POMC and 22-kDa pro-ACTH.
Quantitative real-time PCR Analysis
Quantitative real-time (qRT) PCR was used to quantify anterior pituitary POMC and CRF1-R mRNA and CRF and Brn2 mRNA in the hypothalamus. Total RNA was prepared (Qiagen) per the manufacturer's directions from anterior pituitary glands (n = 5 control, n = 7 LTH) and hypothalami (n = 5 control, n = 7 LTH). Before RT-PCR, total RNA (1 µg) was treated with DNase I (2 U) at 37°C for 60 min; the DNase I was removed via PCR purification columns. Reverse transcription was performed with 1 µg of total RNA per sample, oligo(dT21) as the primer, and Superscript II (Invitrogen) as reverse transcriptase. The details of the reverse transcription reaction have been described previously (23). In addition, a control reaction was performed on each RNA sample in which the reverse transcriptase was omitted to serve as a control for any possible DNA carryover after the DNase I step.
Real-time PCR was performed with cDNA generated from the first-strand synthesis reaction. All PCRs were performed in triplicate. Initial RT-PCRs were performed to ascertain that the amount of cDNA utilized per PCR was within the linear amplification range (POMC, 50 ng; CRF1-R, 200 ng; CRF, 50 ng; Brn2, 50 ng; cyclophilin, 50 ng of cDNA based on amount of RNA in the first-strand reaction). For each primer set (Table 1), the amplicon was subcloned into the TA cloning vector (Invitrogen) and subjected to Sanger dideoxysequencing (Oklahoma Medical Research Foundation Sequencing Core, Oklahoma City, OK) to identify the gene as the correct product. For each primer set, a PCR reaction was also performed on each individual sample from the control (no reverse transcriptase) cDNA reaction to ascertain the dependence of the PCR reaction on cDNA. SYBR Green (1x SYBR Green master mix; Bio-Rad) was utilized as the fluorophore for the qRT-PCR. Real-time PCR was performed utilizing a Bio-Rad iCycler equipped with the real-time optical fluorescent detection system. A three-step PCR was used: an initial denaturation step of 95°C for 10 min to activate the hot-start Taq DNA polymerase followed by sequential cycles consisting of denaturation at 95°C for 45 s, annealing at 55°C for 30 s, and extension at 72°C for 45 s. A total of 35 PCR cycles were performed. A melt curve analysis was conducted on each sample after the final cycle to ensure that a single product was attained; agarose gel electrophoresis confirmed that a single PCR product was of the predicted length. qRT-PCR was performed for each sample (in triplicate) for cyclophilin as a control mRNA by use of the identical first-strand cDNA used for quantification of mRNA for the gene of interest (POMC, CRF1-R, CRF, and Brn2) and in the same PCR run as for the gene of interest to circumvent any between-run variation. Cyclophilin was used as a "housekeeping" mRNA because we previously found that cyclophilin mRNA is not glucocorticoid responsive and does not change in expression in ovine anterior pituitary or adrenal cells in vitro in response to a variety of stimuli (D. Myers, unpublished observations). For quantification purposes, a synthetic single-stranded DNA standard was used to generate a standard curve for extrapolation of starting cDNA concentrations per reaction using the threshold at which the fluorescence of each PCR reaction increased above baseline values for standards to create a linear standard curve (100, 10, 1, 0.1, 0.01, 0.001 pg standard cDNA). The standards were also run in triplicate and with the unknowns in the same PCR block. Extrapolation of unknowns from the standard curve was performed with Prism 4 (Graph Pad Software, San Diego, CA), predicting unknowns from the standard curve threshold values.
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Fetal plasma ACTH1–39, ACTH precursors, and ratios of anterior pituitary ACTH to ACTH precursors between normoxic control and LTH fetuses were compared using a Student's t-test. POMC, CRF1-R, CRF, and Brn2 mRNA were also analyzed using a Student's t-test. All results are expressed as means ± SE.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Two major higher molecular weight ACTH-containing precursors were observed in the anterior pituitary of both control and LTH fetal sheep (Fig. 1A) that comigrated with recombinant ovine POMC (32–35 kDa) and 22 pro-ACTH (22 kDa). We also routinely observed a 17-kDa band consistent with nonglycosylated pro-ACTH (144 residues). A longer exposure of the membrane to film was needed to visualize ACTH (4–5 kDa, Fig. 1B). All molecular weight forms of IR-ACTH were sensitive to competition by preincubating the horseradish peroxidase-labeled ACTH monoclonal antibody used for Western blot analysis with ACTH. In LTH anterior pituitaries, levels of POMC, 22-kDa pro-ACTH (Fig. 2A), and ACTH1–39 (Fig. 2B) were significantly lower compared with the normoxic control fetuses.
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Basal fetal plasma concentrations of ACTH1–39 were significantly elevated in LTH fetuses compared with controls (Fig. 3A). Plasma concentrations of ACTH precursors (POMC and 22-kDa pro-ACTH) were also significantly elevated in LTH compared with control fetuses (Fig. 3B). The ratio of fetal plasma ACTH precursors (POMC and 22-kDa pro-ACTH) to ACTH was significantly lower in the LTH fetal sheep compared with the normoxic control fetuses (Fig. 3C).
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Anterior pituitary concentrations of POMC mRNA were not different between LTH and normoxic control fetuses (Fig. 4A). In contrast, CRF1-R mRNA was significantly increased (P < 0.05) in the LTH group compared with controls (Fig. 4B). Anterior pituitary concentrations of cyclophilin mRNA were not different between the two groups (Fig. 4C).
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There was a trend toward higher CRF mRNA levels in the hypothalami from the LTH fetuses compared with control, but the difference did not reach statistical significance (P = 0.067, Fig. 5A). Brn2 mRNA levels were not different between LTH and control fetuses (Fig. 5B). Brn2 is a POU-domain transcription factor essential for the expression and terminal differentiation of paraventricular nucleus (PVN) neurons and in particular CRF neurons (26). Brn2 has previously been shown to be expressed constitutively and is unresponsive to stress (16). Thus, to control for any differences in dissection of hypothalami between fetuses, we used Brn2 to ascertain inclusion of PVN in the samples.
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LTH fetal anterior pituitaries exhibited a significant reduction (P < 0.03) in CRF1-R protein compared with control fetuses (Fig. 6).
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DISCUSSION |
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In the present study, we found that the basal concentrations of ACTH in the plasma of LTH fetuses were significantly greater compared with that shown in the normoxic controls. In addition, although basal concentrations of ACTH precursors were also elevated in the plasma of LTH fetuses, the ratio of ACTH precursor to ACTH was nonetheless lower in LTH fetal plasma. Because the LTH fetuses have similar basal plasma cortisol concentrations as normoxic age-matched controls (1, 14), the ratio of precursors to ACTH, albeit significantly lower, may not have been sufficient to gain a net increase in the biological activity at the level of the fetal adrenal. However, the results do support that a significant increase in plasma ACTH alone, indeed an approximately fourfold increase, is not sufficient to increase basal plasma cortisol production, thus supporting the concept that ACTH precursors likely function as an important biological regulator of adrenocortical maturation. Another possible explanation for the failure of the LTH fetuses to exhibit increased basal cortisol production despite higher plasma ACTH concentrations and decreased ratio of precursor to ACTH is that the sensitivity of the LTH fetal adrenal to the inhibitory ACTH precursors has increased. Indeed, Schwartz and coworkers (30) showed that only fetal and not adult adrenocortical cells showed diminished ACTH-induced cortisol output by the ACTH precursors.
In the anterior pituitary of the LTH fetuses, POMC mRNA levels exhibited a trend toward being higher, indicating that expression of POMC was being maintained sufficient to sustain POMC production. Both POMC and 22-kDa pro-ACTH levels in the anterior pituitary of the LTH fetal sheep were, however, lower, as was ACTH, indicative of enhanced processing of POMC to ACTH coupled with an enhanced basal release of both the precursors and ACTH. This corresponds with the increase in basal plasma levels of both ACTH and ACTH precursors in the LTH fetal sheep. During late gestation, an increase in the biological activity of IR-ACTH has been observed (8, 25, 34), which suggests a maturation of POMC processing to ACTH1–39. Indeed, the observed shift in the ratio of precursors to ACTH during the final weeks in gestation is mainly due to a selective increase in ACTH as the plasma concentrations of ACTH precursors remain relatively stable.
Although the present study primarily focused on the effect of LTH on POMC processing in the anterior pituitary and changes in circulating ACTH and ACTH precursors, we also addressed potential hypothalamic peptide regulation of the observed changes in anterior pituitary corticotrope function in the LTH fetus. We focused on CRF because delivery of a selective CRF1-R antagonist to fetal sheep has been shown to significantly decrease fetal plasma IR-ACTH concentrations and delay parturition (11). Furthermore, subjecting fetal sheep to hypoxia during late gestation resulted in a specific reduction in the proportion of ACTH-stored CRF target cells in the anterior pituitary (7). Thus CRF appears to be of greater importance relative to AVP in maintaining ACTH biosynthesis and thus adrencortical maturation. In the present study, hypothalamic CRF mRNA showed a trend toward being elevated in the LTH fetal sheep. However, because LTH fetal sheep do not deliver early, it is not surprising that we did not observe a large increase in CRF expression, as this may advance the biological clock governing adrenocortical maturation and not allow the LTH fetus adequate time in utero for maximal development. Perhaps, CRF release was enhanced in the LTH fetal sheep. In support of enhanced CRF release, we did observe an increase in the CRF1-R mRNA in the anterior pituitary of LTH fetal sheep. Furthermore, CRF1-R protein levels in the anterior pituitary of LTH fetuses were decreased compared with levels shown in controls, perhaps as a result of increased internalization because mRNA for the receptor increased. The decreased CRF1-R protein coupled with increased CRF1-R mRNA is consistent with an increased hypothalamic drive-CRF secretion. In support, our group (22) previously showed that lesioning of the fetal PVN results in a decrease in CRF1-R mRNA in the anterior pituitary in conjunction with an increase in CRF binding sites. This likely results from a decreased stimulus for CRF1-R gene expression (lower mRNA) coupled with a decrease in the internalization rate of the receptor (increased binding) upon removal of CRF stimulation as a result of the PVN lesion. Thus the enhanced POMC processing and higher basal levels of ACTH and ACTH-containing peptides in the LTH fetal plasma may reflect increased CRF release in the LTH fetal sheep. However, both CRF and AVP (34) can enhance the biological activity of fetal plasma ACTH and the amount of ACTH relative to ACTH precursors. Thus we cannot exclude a selective increase in AVP expression in medial parvocellular PVN neurons. Our approach for quantifying CRF (qRT-PCR) does not allow for determining AVP levels because magnocellular PVN and supraoptic nuclei expression of AVP would mask any changes in the lower expressing parvocellular PVN neurons.
Stress stimulates ACTH secretion with a subsequent elevation of plasma ACTH concentrations and enhanced adrenal glucocorticoid production (3, 27). The elevation in glucocorticoids leads to a number of catabolic effects, including increased lipolysis, glycogenolysis, and protein catabolism, with a resultant increase in blood glucose levels (20, 31). These changes increase the availability of energy substrates that help the fetus cope with stressful conditions. However, maintenance of elevated glucocorticoid levels for extended periods of time may have deleterious effects. Chronic glucocorticoid exposure can result in a general suppression of anabolic processes. More specifically, it can lead to muscle atrophy and impairment of tissue growth (18–20), elements obviously not favorable to normal fetal growth and development. Additionally, excess cortisol secretion in the sheep fetus could prematurely precipitate parturition. Therefore the ability to "turn off" or neutralize the HPA stress response is of equal importance to the capacity to respond to stress.
The apparent lack of effect of elevated basal plasma ACTH levels on basal cortisol production is perplexing but may reflect the elevation not only in ACTH but also in the ACTH precursors. Indeed, there were 4-fold and 2.5-fold elevations in both ACTH and ACTH precursors, respectively, in the plasma of LTH fetuses compared with the normoxic controls. Furthermore, it is unclear at present whether the inhibitory actions previously observed for the ACTH precursors on cortisol production in ovine fetal adrenocortical cells are strictly competitive with ACTH via the ACTH receptor or through an alternative melanocortin receptor. It was reported that the inhibitory action of the ACTH precursors was on fetal and not adult ovine adrencortical cells (30). Thus the capacity of adrenocortical cells in the LTH fetuses to respond to ACTH and to precursors may also be altered
compared with controls.
To our knowledge, this is the first study to describe the effects of exposure to a long-term (longer than a few weeks) stressor on POMC expression and processing in the ovine fetus. Together, the findings from the present study indicate that the processing of POMC to ACTH has adapted in response to development in a condition of LTH, altering plasma concentrations of ACTH and ACTH precursors and thus maintaining normal fetal adrenocortical maturation under this physiological state. Such information may have important clinical implications.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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