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Endocrinology Vol. 138, No. 8 3435-3443
Copyright © 1997 by The Endocrine Society

ARTICLES

Regulation of Growth Hormone (GH) Gene Expression and Secretion During Pregnancy and Lactation in the Rat: Role of Insulin-Like Growth Factor-I, Somatostatin, and GH-Releasing Hormone1

Javier Escalada, Franco Sánchez-Franco, Beatriz Velasco and Lucinda Cacicedo

Endocrinology Services, Centro de Investigación Clínica, Instituto Carlos III, 28029 (J.E., F.S-F., B.V.), Madrid; and Hospital Ramón y Cajal (L.C.), 28034 Madrid, Spain

Address all correspondence and requests for reprints to: Franco Sánchez Franco, Servicio de Endocrinología, Centro de Investigación Clínica, Instituto Carlos III, C/Sinesio Delgado, 10, 28029 Madrid, Spain.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GH appears to play an important metabolic role during late pregnancy and in lactation maintenance. In this study, pregnant (days 8, 15, and 20 of gestation) and postpartum (days 3 and 8 postpartum, including lactating and nonlactating dams) Wistar rats were used to investigate pituitary GH gene expression and hormone secretion, and the potential alterations of the major signals regulating GH secretion and action [somatostatin (SS) and GH-releasing hormone (GHRH), GH receptor (GH-R), and insulin-like growth factor-I (IGF-I)]. GH and SS messenger RNA (mRNA) were quantitated by Northern blot, and both IGF-I and GH-R mRNA were analyzed by the ribonuclease protection assay technique.

Pituitary IR-GH content and GH mRNA increased at midpregnancy. IR-GH content was decreased in lactating rats. Plasma GH levels progressively increased during pregnancy, whereas no significant alterations were shown during lactation. Elevated GH levels persisted during lactation. Levels at this time were higher in nonsuckling compared with suckling dams. Liver GH-R mRNA progressively decreased during pregnancy, but it remained unchanged during lactation. Plasma IGF-I and liver IR-IGF-I constantly decreased during pregnancy, and no significant modifications were seen either in suckling or in nonsuckling animals. IGF-I mRNA accumulation in the liver decreased during pregnancy. After delivery, a progressive decrease of liver IGF-I mRNA occurred. At the hypothalamic level, a progressive increase in the IR-SS content was found during pregnancy, with no SS mRNA modification. After delivery, a higher hypothalamic IR-SS content was found in lactating than in nonlactating rats, with no changes in SS mRNA levels. Hypothalamic IR-IGF-I also showed a progressive increase during pregnancy with no significant alterations during lactation. Hypothalamic IR-GHRH presented a nonsignificant mild increase during pregnancy with no modifications during lactation. In the pituitary, IR-IGF-I content progressively increased during gestation, reaching its highest concentration at day 20. During lactation, pituitary IGF-I did not change.

In summary, our data show that the mechanisms of the increase in plasma GH levels occurring during pregnancy include an increase in GH gene expression in the pituitary, a decrease in SS secretion from the hypothalamus, an increase in IR-IGF-I content in the hypothalamus and in the pituitary, and a significant decrease in circulating IGF-I. Plasma and liver IR-IGF-I and IGF-I mRNA in the liver decreased throughout gestation due to a lower GH-R gene expression in the liver. This state of GH resistance with a higher GH/IGF-I ratio could be important in providing supplementary nutrients to the fetus. During lactation, GH and its regulatory machinery did not show important modifications.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PREGNANCY and lactation are physiological situations characterized by hormonal changes, such as modifications in plasma gonadal steroids, PRL, and GH concentrations. It has been suggested that GH plays an important role in metabolic changes occurring in late pregnancy (1) and in lactation maintenance in the rat (2). The plasma GH pattern in pregnant and lactating rats is not well understood, although there are data indicating that plasma GH is increased during late gestation. Carlsson et al. (3) demonstrated an increase in basal plasma GH levels during late pregnancy and a marked increase in both plasma GH levels and GH pulse amplitude on day 20 of gestation in the rat, without increase in pulse frequency, but the mechanism is not well understood. In the human, circulating GH levels also increase during pregnancy. The human placenta secretes GH during late pregnancy, resulting in very high serum levels, whereas pituitary GH release appears to be almost completely suppressed (4). This increase in plasma GH is thought to be due to placental expression of a variant human GH (hGH-V) gene. The hGH-V gene product is biologically active (5), and appears to suppress the expression of the normal hGH gene in the pituitary during pregnancy (4). However, the high levels of plasma GH during pregnancy in the rat seem to be derived from the pituitary because hypophysectomy of pregnant rats results in undetectable basal and GH-releasing hormone (GHRH)-stimulated plasma GH levels (3). A pituitary origin of GH secretion in the pregnant rat is also supported by the fact that no counterpart of the hGH-V gene has been found in the rat (6).

During lactation in the rat, the suckling stimulus is known to cause a rapid and transient increase in plasma GH (7, 8), with depletion of pituitary GH concentration (9). A role of hypothalamic GHRH (7) and pituitary ß-endorphin (8) in the control of GH secretion during suckling has been suggested. However, the regulation of pituitary GH gene expression during pregnancy and lactation in the rat is not well documented.

The insulin-like growth factors (IGFs) are polypeptide mitogens that mediate the actions of GH in a variety of tissues. GH-dependent insulin-like growth factor-I (IGF-I) is predominantly expressed and secreted by the liver (10), but the IGF-I gene is also expressed in multiple tissues, including brain and pituitary (11, 12, 13). The IGF-I in rat serum is found mainly associated with a family of IGF binding proteins (IGFBPs). The predominant serum IGFBP is IGFBP-3. During rat pregnancy, maternal serum concentrations of IGF-I decline after day 12 to below nonpregnancy levels (14, 15, 16), and IGFBP-3 becomes undetectable (15, 16, 17). It is known that circulating IGF-I acts on the pituitary to regulate GH synthesis and secretion through inhibitory feedback on the somatotrophs (18, 19). There are also studies indicating that the IGFs might participate in the feedback regulation of GH secretion at the central nervous system (20, 21). Within the hypothalamus, IGF-I has been reported to influence somatostatin (SS) release (18).

The secretion of GH is also under dual hypothalamic inhibitory and stimulatory control. GHRH stimulates GH gene expression and secretion (22, 23), whereas SS inhibits the secretion of GH, modulating the actions of GHRH (24).

The aim of this work was to study the mechanisms implicated in the alterations of GH during pregnancy and lactation and to investigate the changes of the major signals participating in the feedback system of GH regulation, such as plasma IGF-I, the hepatic gene expression and content of IGF-I, the pituitary content of IGF-I, the hypothalamic gene expression and content of SS, and the hypothalamic content of both IGF-I and GHRH during pregnancy and lactation in the rat. We also studied GH receptor (GH-R) gene expression at the hepatic level to investigate the mechanism of IGF-I decrease during rat pregnancy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and experimental design
Adult female Wistar rats (Panlab, Barcelona) were housed in a temperature-controlled room (22 C), with controlled lighting (14 h light, 10 h dark; lights on at 0700 h). Food and water were given ad libitum.

Pregnancy experiment.
Rats were mated during proestrus with male Wistar rats, and the day on which vaginal plugs were observed was designated day 0 of pregnancy. At this time, pregnant females were placed in separate cages. Animals were decapitated at days 8 (n = 5), 15 (n = 7), and 20 (n = 6) of pregnancy.

Lactation experiment.
At the time of delivery (designated day 0 postpartum), rats were randomly assigned to one of two groups: lactating group (L) (litters were adjusted to eight pups for each mother) and control group (C) (all litters were removed). Animals were decapitated at days 3 and 8 of lactation or postpartum (L3, n = 6; C3, n = 6; L8, n = 6; C8, n = 6). Lactation was considered when at least six pups were suckling during 60 min or more.

Rats were decapitated and trunk blood was collected into 5% EDTA tubes. Plasma was stored at -20 C until assayed for immunoreactive estradiol (E2), progesterone (PG), GH, and IGF-I. Hypothalami and pituitaries were removed as has been described (25). For RIA determination, half of each tissue was stored at -80 C until used. For messenger RNA (mRNA) measurement, the other half of each tissue was removed under sterile conditions, rapidly frozen on dry ice, and stored at -80 C until used.

Peptide extraction and immunoassays
For peptide extraction, tissues were homogenized in 1 M acetic acid, boiled for 5 min, and centrifuged (30 min, 4 C). The supernatants were purified in Sep-Pak C18 cartridges, lyophilized, reconstituted in 0.01 N HCl and stored at -20 C until assayed. Plasma E2 levels were measured after dietyl-ether extraction (pregnancy) or directly (lactation) by a commercial RIA (Coat-a-Count Estradiol, DPC, Los Angeles CA). Plasma PG levels were assessed by a commercial RIA (Coat-a-Count Progesterone, DPC). Plasma GH concentrations and pituitary IR-GH content were assessed by a RIA using reagents from NIADDK (Bethesda, MD). Plasma IGF-I levels and liver, hypothalamic, and pituitary IR-IGF-I content were measured by RIA after acid extraction and reverse phase column chromatography using Sep-Pak C18 cartridges (26). This extraction has been demonstrated to completely remove IGF-I binding proteins. A specific antiserum against IGF-I raised in our laboratory (27) was used at a final dilution 1:15,000. Assay sensitivity was 0.25 ng/tube. The intra- and interassay variations were 7% and 15%, respectively. Both hypothalamic IR-SS and IR-GHRH content were measured by previously described RIAs (25, 28) using antisera raised in our laboratory. Before boiling, 50 µl of each sample were taken for total protein determinations by Bradford’s method (29), using BSA as standard. All samples from the same experiment were measured in the same assay, and all the comparisons were made among samples from the same assay.

RNA extraction and measurement of GH, SS, GH-R, and IGF-I mRNA
Total RNA was extracted by Chomczynski’s method (guanidine-phenol-chloroform method) (30). The quality of the RNA was monitored by the ratio of UV absorbance at 260 and 280 nm. A260/A280 ratios were consistently 1.9–2.0, indicating that the samples were essentially free of contaminating proteins. Samples were kept at -80 C until assayed.

GH and SS mRNA analysis
Northern blot analysis for the presence of GH and SS mRNAs was done as follows. Total RNA (2–10 µg) was electrophoresed in 1% agarose-formaldehyde gels, followed by electrotransfer to nylon membranes (Nytran, Schleicher & Schuell, Keene, NH) and UV cross-linking (Hoefer Scientific Instrument, San Francisco, CA). For GH mRNA analysis, the rat GH complementary DNA (cDNA) probe used was the HindIII fragment of the plasmid p-rGH1 (31). The probe was labeled with [32P]deoxy-cytidine 5'-triphosphate (3000 Ci/mmol, Amersham, Arlington Heights, IL) by random primer extension (Boehringer Mannheim, Indianapolis, IN) according to the protocol provided by the manufacturer. Levels of SS mRNA were quantitated by Northern blot hybridization using an antisense SS RNA probe. The antisense probe was constructed by inserting a fragment of the rat preproSS cDNA (32) into the expression vector pSP 65 (Promega Biotech, Madison, WI). The vector containing the SS cDNA insert was linearized with SalI, and a 32P-labeled antisense RNA probe produced with an SP6 polymerase (33). Labeled antisense SS RNA probe was prepared according to the method recommended by the manufacturer (Boehringer Mannheim) using [32P]uridine triphosphate (UTP) (800 Ci/mmol) as radionucleotide. All these procedures were performed under high stringency conditions (33, 34), with 50% formamide at 42 C for GH and at 65 C for SS. Autoradiograms were carried out at -80 C with double high-speed intensifying screens using Kodak X-Omat films (Eastman Kodak Co., Rochester, NY). Equal loading was confirmed by comparing intensities of ethidium bromide-stained ribosomal 28S RNA in the nylon filter. The intensities of bromide-stained filters and autoradiogram signal levels were quantified by densitometry, and expressed as arbitrary units after being normalized for 28S ribosomal RNA (rRNA) levels.

IGF-I and GH-R hybridizations
IGF-I and GH-R mRNA were measured by ribonuclease protection assay (RPA). For IGF-I mRNA analysis, a rat cDNA IGF-I probe inserted in the pGEM-3 plasmid was used and was HindIII linearized (35). An antisense complementary RNA (cRNA) was constructed using 32P-UTP and a T7 RNA polymerase. The RPA showed two major protected fragments of 376 bp (IGF-Ib) and 224 bp (IGF-Ia). Identical amounts of total RNA (liver: 5 µg; pituitary: 10 µg; hypothalamus: 15 µg) of each experimental group were hybridized at 45 C, for 14–18 h. The hybridization solution contained 75% formamide, 80 mM Tris HCl, pH 7.6, 4 mM EDTA, 1.6 M NaCl, 0.4% SDS and IGF-I probe (5 x 105 cpm/sample). After the hybridization, samples were digested using RNAasa-A (40 mg/ml) and RNAasa-T1 (2 mg/ml), phenol-chloroform extracted, and ethanol precipitated. Then, samples were electrophoresed on a denaturalizing gel (8% polyacrilamide/8 M urea). Autoradiographies of gels were carried out at -80 C with double high-speed intensifying screens using Kodak X-Omat films. The intensities of autoradiogram signal levels were quantified by densitometry, and expressed as arbitrary units. Both IGF-I mRNA subspecies (IGF-Ib and IGF-Ia) showed similar changes. Therefore, only IGF-Ia was densitometrically analyzed, and the densitometric reading of each sample expressed as arbitrary units.

For GH-R mRNA analysis, a rat cDNA GH-R probe inserted in the pT7T3 18U plasmid was used and was BamHI linearized (36). An antisense cRNA was constructed using 32P-UTP and a T7 RNA polymerase. This probe produces two protected bands when hybridized to total liver RNA: a 439-base band corresponding to the GH-R mRNA, and a 298-base band corresponding to the alternately spliced mRNA which, in the rat, encodes the GH-binding protein (37). The hybridization solution was identical to that used for IGF-I mRNA analysis. Autoradiographies of gels were carried out at -80 C with double high-speed intensifying screens using Kodak X-Omat films. Equal loading was confirmed by cyclophilin. The cyclophilin probe was a full-length rat cDNA inserted in the pGEM-5Zf(-) plasmid and was ApaI linearized. An antisense cRNA was constructed using 32P-UTP and an SP6 RNA polymerase, as has been described (38). The hybridization solution was identical to that described for IGF-I mRNA. The intensities of autoradiogram signal levels were quantified by densitometry, and expressed as arbitrary units after being normalized for cyclophilin levels.

The values represent the mean ± SEM of three quantitations of pools from two animals (three repetitions). Tissues from all animals were analyzed following the same procedure for the GH-R and IGF-I determinations.

Statistical analysis
All the quantifications were performed at least twice. Data were analyzed by ANOVA followed by the Student-Newmann-Keul’s test for multiple comparisons between groups. A P value of less than 0.05 was considered to be significant. Results are expressed as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Plasma gonadal steroids during pregnancy and lactation
As shown in Table 1Go, plasma E2 levels increased throughout gestation and reached their maximum value on day 20 of the study, 1 day before delivery. On the contrary, as can be seen in Table 2Go, very low values of E2 were found during lactation, with similar values on days 3 and 8 in the suckling rats. The plasma E2 was significantly lower in the lactating rats than in the nonlactating animals (P < 0.01).


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  		Table 1. Circulating gonadal steroid levels during pregnancy

 

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  		Table 2. Circulating gonadal steroid levels in lactating and in nonlactating control rats

 
Plasma PG progressively increased up to day 15 of pregnancy and decreased thereafter until day 20 (P < 0.01 vs. days 8 and 15, Table 1Go). During lactation, plasma PG levels were higher (P < 0.01) in suckling rats than in their corresponding controls, and increased significantly throughout lactation (Table 2Go).

Plasma GH levels in pregnant and postpartum rats
During pregnancy, plasma GH levels increased up to day 15 of gestation, and remained at this level on day 20 (day 8: 20.9 ± 2.2 ng/ml; day 15: 62 ± 9.3; day 20: 71.3 ± 14.3) (Fig. 1AGo). After delivery, plasma GH went down to normal levels, and no changes were found either in lactating or nonlactating rats (Fig. 1BGo).



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  		Figure 1. A, Plasma GH levels in pregnant rats. During pregnancy, plasma GH progressively increased up to day 20 of gestation. P8, day 8 of pregnancy; P15, day 15 of pregnancy; P20, day 20 of pregnancy. *, P < 0.05 vs. P8. B, Plasma GH levels in lactating rats. No changes were found either during lactation or in nonsuckling control rats. L3, day 3 of lactation; C3, control nonsuckling rats on day 3 of postpartum; L8, day 8 of lactation; C8, control nonsuckling rats on day 8 of postpartum. Values represent mean ± SEM of six animals.

 
Pituitary GH mRNA levels and peptide content during pregnancy and lactation
For a better understanding of the mechanisms of plasma GH alterations, pituitary IR-GH content and GH mRNA levels were studied. Pituitary IR-GH content increased at midpregnancy (P < 0.05, Fig. 2AGo), and declined slightly by late pregnancy. During lactation, the content of pituitary IR-GH was lower in lactating rats than in control nonlactating dams on both days 3 and 8 (Fig. 2BGo).



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  		Figure 2. A, Pituitary IR-GH content during rat pregnancy. Pituitary IR-GH concentrations were higher on late pregnancy when compared with early pregnancy (P8, day 8 of pregnancy; P15, day 15 of pregnancy; P20, day 20 of pregnancy). *, P < 0.05 vs. P8. B, Effect of lactation on pituitary IR-GH content in comparison with nonsuckling control rats. During lactation, content of pituitary IR-GH was lower in lactating rats when compared with control rats (L3, day 3 of lactation; C3, control nonsuckling rats on day 3 of postpartum; L8, day 8 of lactation; C8, control nonsuckling rats on day 8 of postpartum). *(a), P < 0.05 vs. L3; *(b), P < 0.05 vs. L8. Values represent mean ± SEM of six animals. C, Pituitary GH mRNA level during rat pregnancy. Higher GH mRNA accumulation was found on late pregnancy in comparison with early pregnancy (*, P < 0.05). Figure represents GH mRNA of 0.8 kilobases (top), etidium bromide signal of 28S rRNA as a load control (middle), and densitometric reading of GH mRNA expressed as arbitrary units after normalizing for 28S rRNA levels (bottom). Values represent mean ± SEM of three quantitations of pools from two animals (three repetitions). D, Effect of lactation on pituitary GH mRNA in comparison to control nonsuckling rats.

 
Northern blot analysis of pituitary GH mRNA (Fig. 2CGo) showed a pattern similar to pituitary IR-GH content, indicating a potential transcriptional mechanism of the GH increase in the pituitary. Pituitary GH gene expression was not altered during lactation (Fig. 2DGo).

Liver GH-R mRNA accumulation in pregnant and lactating rats
To understand the mechanism of GH alterations during these physiological conditions, GH-R mRNA was quantitated by RPA in liver extracts. Two different transcripts were obtained, corresponding to GH-R mRNA (439 bp) and GH binding protein (GHBP) mRNA (290 bp). GH-receptor mRNA levels decreased at day 15 of gestation and remained at the same level on day 20 (Fig. 3AGo). After delivery, GH-R mRNA accumulation in the liver showed no significant alterations in the lactating animals but there was an increase in the controls on the third day postpartum (Fig. 3BGo). GHBP mRNA was not altered either in pregnant rats or lactating rats (Fig. 3Go, C and D).



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  		Figure 3. A, Liver GH-R and GHBP mRNA accumulation during rat pregnancy quantitated by RPA. A decrease from days 8–20 of gestation was observed. *, P < 0.05. Lanes were: 1, P8; 2, P15; 3, P20; 4, L3; 5, C3; 6, L8; 7, C8; 8, PBR322 (molecular weight control); 9, nondigested cyclophilin riboprobe; 10, nondigested GHR/GHBP riboprobe (positive control); 11, GHR/GHBP and cyclophilin riboprobes digested with RNase (negative control). Values represent mean ± SEM of three quantitations of pools from two animals (three repetitions). B, Liver GH-R mRNA levels during lactation. A decrease was observed at day 8 postpartum. C, Liver GHBP mRNA levels during rat pregnancy quantitated by RPA. No changes were observed throughout gestation. D, Liver GHBP mRNA levels during lactation. No significant differences were observed.

 
Plasma IGF-I levels during pregnancy and lactation
According to the decrease of GH-R gene expression in the liver, Fig. 4AGo shows that circulating IGF-I decreased significantly between days 8 and 15 of gestation (100.4 ± 3.2 ng/ml and 84.3 ± 4.7, respectively; P < 0.05), and reached its lowest value on day 20 (57 ± 1.5; P < 0.001 vs. day 8 and P < 0.01 vs. day 15). Plasma IGF-I levels showed no significantly different values between lactating and control rats, and levels similar to early pregnancy were observed (Fig. 4BGo).



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  		Figure 4. A, Plasma IGF-I levels during pregnancy. Circulating IGF-I concentrations decreased throughout gestation, reaching their lowest value on day 20. *, P < 0.05 vs. P15; **, P < 0.01 vs. P20; ***, P < 0.001 vs. P20. B, Plasma IGF-I levels during lactation. No significant modifications of plasma IGF-I were observed during lactation in comparison with nonsuckling control rats. Values represent mean ± SEM of six animals.

 
Liver IGF-I mRNA and IR-IGF-I concentrations in pregnant and lactating rats
The pattern of IR-IGF-I concentrations in the liver of pregnant rats was similar to that described for plasma IGF-I (Fig. 5AGo). Liver IGF-I concentration decreased on day 15 (17.7 ± 1.1; P < 0.05 vs. day 8) and reached the minimum value on day 20 (13.3 ± 1; P < 0.01 vs. day 8). Liver IR-IGF-I values correlated well with plasma IGF-I levels (r = 0.57, P < 0.05). During lactation, liver IR-IGF-I concentrations were not altered, as has been shown for plasma IGF-I levels (Fig. 5BGo).



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  		Figure 5. A, Liver IR-IGF-I concentration in pregnant rats. Hepatic content of IR-IGF-I decreased from days 8–20 of gestation. *, P < 0.05 vs. P15; **, P < 0.01 vs. P20. B, Liver IR-IGF-I content in lactating rats. No significant changes in liver IGF-I concentrations were seen during lactation. Values represent mean ± SEM of six animals.

 
Liver IGF-I mRNA was studied to determine whether the decline in both plasma and liver IR-IGF-I concentrations in rats during late pregnancy coincided with a decreased abundance of liver IGF-I message. The RPA showed two major IGF-I transcripts with estimated sizes of 376 bp (IGF-Ib) and 224 bp (IGF-Ia) (Fig. 6Go top and 6). During pregnancy, the abundance of IGF-Ia mRNA decreased on day 20 of gestation (P < 0.05 vs. P8 and P15; Fig. 6CGo);. The accumulation of IGF-I mRNA decreased from days 3–8 of lactation and reached similar concentrations to control rats on days 3 and 8 of lactation (Fig. 5DGo).



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  		Figure 6. A and B, Two major IGF-I mRNA species quantitated by RPA (top) and densitometric reading of liver IGF-Ia mRNA (bottom). Lanes were: 1, P8; 2, P15; 3, P20; 4, L3; 5, C3; 6, L8; 7, C8; 8, PBR322 (molecular weight control); 9, nondigested cyclophilin riboprobe; 10, nondigested IGF-I riboprobe (positive control); 11, IGF-I and cyclophilin riboprobes digested with RNase (negative control). Values represent mean ± SEM of three quantitations of pools from two animals (three repetitions). C, Liver IGF-I mRNA content during rat pregnancy. Quantitation by RPA showed two major IGF-I mRNA species (IGF-Ib of 376 bp and IGF-Ia of 224 bp). During pregnancy, abundance of liver IGF-I mRNA (IGF-Ia) decreased at day 20 of gestation. *, P < 0.05. D, Liver IGF-I mRNA accumulation in lactating rats. Levels of IGF-I mRNA decreased throughout lactation, and on day 8 reached similar concentrations to nonsuckling control rats. *(a), P < 0.05 (L8 vs. L3); *(b), P < 0.05 (C8 vs. C3).

 
Hypothalamic SS mRNA levels and IR-SS concentrations during pregnancy and lactation
The additional participation of hypothalamic SS in GH regulation during pregnancy and lactation was studied. The IR-SS concentrations in the hypothalami of pregnant rats increased throughout gestation, and was statistically significant on day 20 of pregnancy (Fig. 7AGo). As shown in Fig. 7BGo, hypothalamic IR-SS content was higher (P < 0.01) in the lactating rats than in their respective nonsuckling control animals, which recovered similar values to those seen in early pregnancy soon after delivery (C3, Fig. 7BGo).



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  		Figure 7. A, Hypothalamic IR-SS concentration during pregnancy. IR-SS content increased in hypothalamus of pregnant rats, reaching its highest value on day 20 of gestation. **, P < 0.01 vs. P8 and P15. B, Hypothalamic IR-SS concentration in lactating and nonlactating control rats. Hypothalamic IR-SS content was higher in lactating rats than in their respective controls. ** (a), P < 0.01 vs. C3; ** (b), P < 0.01 vs. C8; ** (c), P < 0.01 vs. L8. Values represent mean ± SEM of six animals. C, Hypothalamic SS mRNA quantitated by Northern blot during pregnancy. No significant differences were found. D, Hypothalamic SS mRNA during lactation. Northern blot analysis of SS mRNA showed no change during lactation. C and D, SS mRNA of 0.6 kilobases (top), etidium bromide signal of 28S rRNA as a load control (middle), and densitometric reading of SS mRNA expressed as arbitrary units after normalizing for 28S rRNA levels (bottom). Values represent mean ± SEM of three quantitations of pools from two animals (three repetitions).

 
Northern blot analysis of SS mRNA showed no changes during pregnancy (Fig. 7CGo) or in lactating and control mothers (Fig. 7DGo).

Hypothalamic IR-GHRH content during pregnancy and lactation
It is accepted that GHRH stimulates GH gene expression and GH secretion. Therefore, its potential participation in GH changes during pregnancy and lactation was studied. Although an upward trend in hypothalamic IR-GHRH concentration during pregnancy was detected, there was no significant modification of hypothalamic IR-GHRH content either in pregnant (Fig. 8AGo), or in lactating and control rats (Fig. 8BGo) at any time during the study, probably due to the great dispersion of the data.



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  		Figure 8. A, Hypothalamic IR-GHRH concentration during pregnancy. No significant changes of hypothalamic IR-GHRH were shown. B, Hypothalamic IR-GHRH concentration in lactating and nonlactating control rats. During lactation no differences were found in hypothalamic IR-GRF content between groups. Values represent mean ± SEM of six animals.

 
Hypothalamic IR-IGF-I content during pregnancy and lactation
The IR-IGF-I concentration in the hypothalamus of pregnant rats was higher at late pregnancy as compared with the early gestation, reaching its highest value on days 15 and 20 (P < 0.05 vs. P8; Fig. 9AGo). During lactation no differences were found in the hypothalamic IR-IGF-I content among the groups (Fig. 9BGo).



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  		Figure 9. A, Hypothalamic IR-IGF-I concentration in pregnant rats. Hypothalamic content of IGF-I increased on days 15 and 20 of pregnancy. *, P < 0.05 vs. P8. B, Hypothalamic IR-IGF-I concentration in lactating and nonlactating control rats. There were no significant modifications either in lactating or in control rats. Values represent mean ± SEM of six animals.

 
Pituitary IR-IGF-I content during pregnancy and lactation
The pituitary IR-IGF-I content in pregnant rats showed a pattern similar to that described for hypothalamic IR-IGF-I. Pituitary IR-IGF-I concentrations increased throughout gestation and reached their highest values on day 20 (P < 0.001 vs. day 8 and 15) (Fig. 10AGo). During lactation, pituitary IR-IGF-I content did not show modifications (Fig. 10BGo).



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  		Figure 10. A, Pituitary IR-IGF-I concentration during pregnancy. Pituitary IGF-I content increased throughout gestation, reaching its highest value on day 20. ***, P < 0.001 vs. P8 and P15. B, Pituitary IR-IGF-I concentration in lactating and nonlactating control rats. During lactation, pituitary IR-IGF-I did not change. Values represent mean ± SEM of six animals.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In agreement with other authors (3), this study confirms that GH levels in the rat plasma increase during mid and late pregnancy, represented by day 15 and day 20 of gestation. This increment has been attributed to a longer half-life of GH (39). However, our data indicate that the high plasma GH levels of pregnancy may be of pituitary origin, because they coincide with higher pituitary IR-GH content and GH mRNA accumulation. Therefore, a transcriptional mechanism increasing GH gene expression in the pituitary seems to be activated on late pregnancy. A pituitary origin of the increased GH levels in the plasma of pregnant rats has also been suggested in other studies that used different methods (3, 6). However, in human pregnancy, the increased plasma GH levels are suggested to be secondary to placental expression of hGH-V gene (4).

During lactation, plasma GH levels did not change at any time in the study and showed similar values to nonsuckling control rats. Apparently, this is not in agreement with other authors who showed a rapid and transient increase in serum GH concentrations after the suckling stimulus, and a return to baseline values by 30–60 min after the onset of suckling (7, 8). A possible cause of this discrepancy could be the differences in the experimental design used in this study, as the animals were sacrificed at least 1 hour after the onset of the suckling stimulus. Therefore, it is possible that the rapid and transient increase in plasma GH levels could have disappeared by that time. At the pituitary level, we found lower IR-GH concentrations in lactating rats when compared with control dams, with no alterations in GH mRNA accumulation. This may suggest, as Grosvenor et al. (9) demonstrated, that the suckling stimulus induces a depletion of pituitary concentration of GH in the lactating rat, consequently the rapid and transient increase of plasma GH levels induced by the suckling stimulus may be due to signal(s) that increase GH secretion, but do not act at the transcriptional level.

The data shown in this study demonstrate that the hepatic GH-R mRNA decreased throughout gestation, whereas liver GHBP mRNA levels were similar at late pregnancy and lactation. Plasma IGF-I levels and liver IR-IGF-I content continuously decrease along pregnancy. Moreover, lower IGF-I mRNA levels were found in the liver on day 20 of pregnancy than on the other days of this study. Consequently, this confirms other reports using Northern blot analysis (14, 15). These results indicate that one mechanism for the reduction of plasma IGF-I levels during late pregnancy is a transcriptionally regulated decrease in hepatic synthesis of IGF-I, at least on day 20 of pregnancy. The mechanisms operating in the decrease of plasma IGF-I and liver IR-IGF-I content on day 15 of pregnancy seem to be different as a concomitant decrease in liver IGF-I mRNA was not observed. The decrease of plasma IGF-I during mid and late pregnancy in the rat could be the consequence of a coincidental decrease of IGFBP-3 (15, 16), leading to the decrease in the half-life of serum IGF-I. In fact, Davenport et al. (15) demonstrated that IGFBP-3 is degraded by one or more proteases that are present in the serum of pregnant rats, and no changes in hepatic IGFBP-3 mRNA were demonstrated (40). Much data from previous studies suggest a placental origin of the substances causing the decrease in IGFBP-3 (41).

The depletion of liver IGF-I has been attributed to a state of GH resistance (14, 16), but the mechanisms are still a controversial issue. Travers et al. (14) demonstrated that GH normally binds to its liver receptor during pregnancy. Mathews et al. (36) showed that the levels of hepatic GH-R mRNA are indeed increased during pregnancy, whereas Tiong and Herington (42) found that only the truncated GHBP mRNA was up-regulated, and they also observed no changes in the full-length GH-R mRNA. Based on these results, it has been proposed that the GH resistance appearing during late pregnancy occurs at a postreceptor level (14). However, in this study, liver GH-R mRNA decreased throughout gestation, reaching its lowest value on day 20, whereas GHBP mRNA levels remained unchanged. The discrepancies with other authors of the GH-R mRNA results could be due to methodological differences. We analyzed GH-R mRNA levels on precise days throughout pregnancy, whereas other studies only considered a pool of days from 18–21 of pregnancy. Consequently, our data allow us to postulate that the GH resistant state of mid and late pregnancy occurs at the GH-R level, without excluding additional postreceptor mechanisms not studied in these experiments. Although the factors implicated in this state of GH resistance are not known, the high circulating E2 levels of pregnancy could play a role. Estrogens have been shown to suppress circulating IGF-I and enhance GH secretion in postmenopausal women with oral ethinyl estradiol treatment (43), and to inhibit hepatic IGF-I mRNA generation in the rat (44). Furthermore, the circulating concentrations of rat placental lactogens that change considerably during the period of gestation could be implicated in this state of GH resistance.

The mechanisms controlling GH secretion during pregnancy and lactation of the rat remain unknown. It has been suggested that the GH peaks in pregnant rats are due to concomitant release of GHRH (3), as previously suggested for nonpregnant female rats (45). Although GHRH has been detected in the rat placenta (46), the importance of placental GHRH for the increase in plasma GH levels during gestation is unknown. With regard to the implication of circulating IGF-I in GH regulation, we have shown in this study, and other authors have also reported, that plasma levels of IGF-I dramatically decrease during mid and late pregnancy in the rat (14, 15, 16), and there are data supporting that circulating IGF-I inhibits pituitary GH synthesis and secretion acting on the somatotrophs (18, 19). Therefore, it can be accepted that diminished negative feedback as a consequence of the decrease in IGF-I may contribute to the increase of plasma GH during late pregnancy.

The implication of the hypothalamus in the regulation of GH during pregnancy and lactation was also studied. There was an increase in IR-SS content during pregnancy, mainly on day 20, but no changes in SS mRNA levels were found. These data could be interpreted as the result of an inhibition of hypothalamic SS secretion that occurs in late pregnancy, and therefore could participate in the increase of plasma GH. This decline in SS release could be accounted for by a decline in circulating IGF-I, because it is known that IGF-I administration increases hypothalamic SS (18). To a certain extent, a situation of GH-R resistance might be involved and account for the increments on hypothalamic SS. In fact, previous studies have shown that GH excess leads to an increase in SS release in vitro from normal hypothalami (47). However, a prolonged GH-R resistance might also affect SS gene expression, and our data indicate that SS mRNA is not altered during pregnancy. With regard to GHRH, no significant changes in hypothalamic IR-GHRH content were observed during gestation, although there was a trend toward increasing levels on late pregnancy.

Within the central nervous system, high levels of IGF-I have been detected in the rat hypothalamus as well as in the anterior pituitary (11, 12, 13). The presence of both IGF-I mRNA (12) and IGF-I receptor mRNA (48) in the rat brain indicates active synthesis and potential biological activity of these peptides within the central nervous system. Thus, we studied the IR-IGF-I content at the hypothalamic and pituitary level during pregnancy. Our data show that hypothalamic IR-IGF-I concentrations, which are decreased at early pregnancy as compared with the postpartum period, are recovered during late pregnancy. Therefore, an inhibition of IGF-I release appears to occur during late pregnancy. It has been shown that estrogens up-regulate 125I-IGF-I binding and IGFBP2 in the pituitary (49), and so the possibility also exits that during pregnancy more IGF-I being extracted from the circulation and sequestered in the pituitary and the hypothalamus. As has been previously shown for SS, the decrease of hypothalamic IGF-I secretion could be implicated in the increase of plasma GH levels at this stage of gestation. In fact, several in vivo data have suggested that the intracerebroventricular administration of a preparation enriched in both IGF-I and IGF-II (20) or of IGF-I alone (21) causes a marked suppression of the spontaneous pulses of GH release in the rat, indicating that the IGFs might participate in the feedback regulation of GH secretion at the central nervous system level.

Within the hypothalamus, IGF-I has been reported to increase both SS (18) and GHRH (50) release. Our data suggest that IGF-I, the content of which increases in late pregnancy, could lead through paracrine/autocrine mechanisms to the decrease in SS secretion. The complexity of the neuroendocrine regulation for this physiological situation, in which estrogens and binding proteins might be playing a role, makes it difficult to propose a unique regulatory mechanism. During lactation only changes in hypothalamic SS content were found, with higher SS content in lactating than in control rats. The higher content of IR-SS during lactation was not accompanied by changes in the SS gene expression, suggesting an inhibition of SS secretion. These data support the fact that the rapid and transient increase in plasma GH levels during the suckling stimulus could be due to an inhibition of hypothalamic SS secretion. The duration time of the low SS tone could be longer that the increase in plasma GH levels. The absence of changes in plasma, liver, hypothalamic, and pituitary IGF-I during lactation suggests that this peptide does not play an important role in the GH regulation in the suckling rat.

During pregnancy, we found that pituitary IR-IGF-I content progressively increased, reaching its highest concentration on day 20. The increment in pituitary IGF-I could also participate in the increase of plasma GH levels occurring during late pregnancy. These data also show the tissue-specific regulation of IGF-I gene expression during pregnancy in the rat. In fact, pituitary IGF-I receptors have been demonstrated (51), consequently both pituitary GH and IGF-I may mutually interact by a paracrine and/or autocrine feedback mechanism (52).

In conclusion, we showed that the increase in plasma GH levels occurring during late pregnancy in the rat is probably regulated at a transcriptional level. This supports a pituitary origin of plasma GH during pregnancy. In contrast, peripheral IGF-I continuously declines during pregnancy, and this decrease is due, at least in part, to a lower hepatic synthesis of IGF-I. The discrepancy between plasma GH and plasma IGF-I levels seems to be secondary to a state of GH resistance due to a decrease in liver GH-R gene expression. We suggest that the secretion of hypothalamic SS and hypothalamic and pituitary IGF-I is inhibited during late pregnancy due to unknown causes. Therefore, these facts, along with the low plasma IGF-I levels at this stage of pregnancy, could be implicated in the increase of plasma GH levels during late pregnancy. The role of GHRH warrants further studies.

During lactation, IGF-I did not show any modification at the times of the study, except at hepatic level, suggesting that it does not play an important role in the GH regulation in the suckling rat. We found lower pituitary IR-GH content and higher hypothalamic IR-SS content in the lactating than in the control rats. Therefore, it is possible to hypothesize that this is the cause for the rapid and transient increase of plasma GH levels described during the suckling stimulus, but this was probably not detected in our study due to the experimental design.


   Acknowledgments
 
The authors thank Dr. Eleuterio Hernández for providing the IGF-I probe. We also thank Mary Harper for the preparation of the manuscript, the National Hormone and Pituitary Distribution Program, NIDDK, for supplying the rat GH RIA reagents, and Purificación Mota for technical assistance.


   Footnotes
 
1 This work was supported by the Beca de Ampliación de Estudios of FIS (92/5107 and 93/5094), grants from DGICYT (PM 90–0029, PM 90–0030), and a grant from Fundación Valgrande. Back

Received September 16, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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