0013.72’27/92/1312-0676$03.00/O Endocrinology Copyright G 1992 by The Endocrine

Vol. 131, No. 2 Printed in LISA.

Society

Expression of the Growth Growth Hormone-Binding the Mouse*

Hormone Protein

SCOTT D. CRAMER, ROSS BARNARD?, AND FRANK TALAMANTESt

ENGBERS,

Department

of Biology, Sinsheimer

Laboratories,

CHARLES University

Receptor and during Pregnancy

LINDA

of California,

in

OGREN,

Santa Cruz, California

95064

ABSTRACT A 20-fold increase in the relative expression of the hepatic GHbinding protein (GHBP)-encoding message between nonpregnant and l7-day pregnant mice was found. The hepatic GH receptor (GHR)encoding message increased &fold between nonpregnant and pregnant mice. The increase in both messages began on day 9 of pregnancy. The steady state level of the GHBP-encoding message continued to increase steadily until day 17 of pregnancy; however, by day 13 of pregnancy, the steady state level of the GHR-encoding message reached a plateau that continued to day 17. The ratio between the GHBPand GHRencoding messages gradually increased during the second half of pregnancy, reaching a maximum on day 17. There was a lo- to 16-fold increase in GH-binding capacity in liver microsomes and a 30- to 50-fold increase in serum GH-binding capacity

between nonpregnant and late pregnant mice. The increase in hepatic GH-binding capacity began on day 9 of pregnancy and reached a plateau on day 11, which was maintained until the end of gestation. The increase in serum GH-binding capacity began on day 9 of pregnancy and continued to increase until day 17. No significant change in mouse (m) GHR (mGHR) or mGHBP affinity constants were observed between nonpregnant and pregnant mice; however, the mGHR had a 20fold greater affinity for mGH than did the mGHBP. The serum GH concentration increased in the second half of pregnancy. The GHBPbound and the free fractions of GH during pregnancy were predicted. While the bound fraction of GH is predicted to parallel the total GH concentration measured by RIA, the concentration of free mouse GH remains unchanged during pregnancy. (Endocrinology 131: 876-882, 1992)

G

pregnancy that may also regulate GHR and GHBP. Examination of the temporal relationship between the rise in GHR/ GHBP mRNA steady state levels and when other endocrine/ physiological changes occur during pregnancy might reveal critical mediators involved in the regulation of GHR/GHBP mRNA steady state levels. This study describesand characterizes the temporal relationship of the steady state mRNA levels for GHR and GHBP in liver and characterizes the hepatic microsomal and serum GH-binding capacitiesduring pregnancy in the mouse.

H REGULATES the growth and differentiation of target tissues by interacting with a membrane-bound GH receptor (GHR). In serum, GH circulates bound to a GHbinding protein (GHBP) that is biochemically (1) and immunologically (2) related to the GHR. In humans and rabbits, it has been speculatedthat the GHBP is produced by proteolytic cleavage of the extracellular domain of the GHR (1). In rats, it has been clearly demonstrated that all of the GHBP is produced by a mRNA that is distinct from GHR mRNA (3, 4). In mice, a separate mRNA has also been identified that is homologous to the rat GHBP-encoding mRNA and is predicted to encode a secretedGHBP (5). During pregnancy in the mouse, there are parallel increasesin serum GH (6), liver GHR (7), and serum GHBP (8). Increaseshave also been demonstrated in liver GHR and GHBP mRNAs in nonpregnant and late pregnant mice (5, 9). In rats, slight increasesin liver GHR, serum GHBP, and liver GHR and GHBP mRNA levels have been reported during pregnancy (10). In humans, a large increasein hepatic microsome GH-binding sites during pregnancy was recently reported (11). The factors regulating GHR and GHBP expression during pregnancy are poorly understood. In mice, GH is thought to be partially responsiblefor the increasein GHR and GHBP mRNA and protein levels (12). Several other endocrine changes,besidesan increase in GH, occur during

Materials and Methods Animals Timed pregnant Swiss Webster mice were obtained from Simonsen Laboratories (Gilroy, CA). The day that a plug was found was considered day 0 of pregnancy. The mice were housed in a controlled environment, with a 14-h light, 10-h dark lighting cycle (lights on, 0600 h) and allowed free access to food and water. The care and use of animals in this study were approved by the University Animal Care Committee.

Hormones

and iodinations

Mouse (m) GH was purified as previously described (13). Recombinant bovine (rb) GH was a generous gift from Monsanto (St. Louis, MO). rhGH was obtained from Genentech (South San Francisco, CA). Hormones were radiolabeled with Na’? (Amersham, Arlington Heights, IL) by the Iodogen method (14). Iodogen was purchased from Pierce (Rockford, IL).

Received March 11, 1992. * This work was supported by NIH Grant DK-42361 (to F.T.). t Supported by the C. J. Martin Fellowship of the National Health and Medical Research Council of Australia. $ To whom requests for reprints should be addressed.

RNA isolation

and Northern

analysis

Whole livers were collected and immediately frozen on dry ice. Total RNA was isolated by the guanidium isothiocyanate-phenol-chloroform

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EXPRESSION

OF GHR

AND

extraction method (15), and total RNA was quantitated by optical density at 260 nm. Liver total RNA was isolated separately from four individuals in each of the following physiological states: nonpregnant females and 5-, 7., 9-, ll-, 13-, 15-, and 17.day pregnant mice. Three males were also used for isolation of liver total RNA. Total RNA was separated on 1.5% agarose-6% formaldehyde gels and transferred to a Nytran nylon membrane (Schleicher and Schuell, Keene, NH) by standard procedures (16). 32P-Labeled cDNA probes were generated using the Multiprime cDNA labeling method (Amersham, Arlington Heights, IL). To probe blots for GHR and GHBP messages, the 190-basepair (bp) 5’ EcoRI/ Hind111 fragment of clone GHR/BP.23 was used (5). This fragment contains 89 bp of 5’-untranslated sequence, followed by the first 101 bp of coding sequence of both the GHR and GHBP mRNAs. This fragment has previously been shown to hybridize to both the 1.2- and 3.9-kilobase (kb) GHR/GHBP messages (5). To control for RNA sample loading and transfer efficiency differences, the blots were probed with the 0.9-kb cDNA for the human ribosomal protein gene L7/pHE-7 (L7). The abundance of the L7 message has been shown to remain constant under many different physiological conditions (17-20). Hybridizations were conducted at 42 C for 16 h in 50% formamide, 5 X SSPE [20 X = 3.6 M NaCl, 0.2 M NaP04 (pH 7.7), and 20 mM EDTA], 5 X Denhardt’s reagent [l x Denhardt’s = 0.02% (wt/vol) Ficoll, 0.02% (wt/vol) polyvinylpyrrolidone, and 0.02% (wt/vol) BSA], 0.1% (wt/vol) sodium dodecyl sulfate (SDS), and 50 fig/ml sheared salmon sperm DNA. Blots were washed twice at 22 C for 20 min each time in 5 X SSPE-0.1% SDS, twice at 42 C for 20 min each time in 0.2 X SSPE-0.1% SDS, and twice at 53 C for 20 min each time in 0.2 X SSPE-0.1% SDS. Kodak XAR5 x-ray film (Eastman Kodak, Rochester, NY) was exposed to blots at -70 C until the film was develoued. All blots were hvbridized and washed at the same time and in the ‘same hybridization/washing vessel. Exposure and development of x-ray film were carried out at the same time.

Densitometry

analysis

Autoradiographs were analyzed by transmittance densitometry on a Bio-Rad (Richmond, CA) model 620 video densitometer. Densitometric images were analyzed using the 1D Analyst II (V.3.10) software from Bio-Rad. Scanning and image analysis parameters were maintained constant for all autoradiographs. Background noise was determined independently for each autoradiograph and subtracted from each scan before calculation of optical density. One lane of each gel was loaded with the same amount (-5 pg) of a pool of liver total RNA from four 17-day pregnant mice to control for differences in RNA loading between gels. The peak OD height for this lane was arbitrarily assigned a value of 1. The peak OD heights for L7 message from all lanes on the autoradiograph were divided by the peak OD height for L7 message from the lane loaded with the 17-day pregnant pool in order to determine the relative RNA loading on the gel. The relative loading values were then used to adjust the peak OD height values from the autoradiographs of the blots probed with the 5’ EcoRI/HindIII fragment of clone GHR/BP.23. This approach of using the same quantity of the same sample (17-day pregnant pool) in multiple Northern blots allowed for both normalization within a given autoradiograph and normalization between autoradiographs.

Tissue collection and membrane

preparation

Blood was collected from mice as previously described by this laboratory (21). The blood was allowed to clot on ice for 30 min. The samples were centrifuged at 4,000 X g for 15 min at 4 C. The serum fraction was collected and stored at -70 C until use. Livers were dissected from animals immediately after death, frozen on dry ice, and stored at -70 C until use. Microsomal fractions were prepared from individual livers, as previously described (8). Tissue was homogenized with a Brinkmann Polytron homogenizer (Brinkmann Instruments, Westbury, NY) in 2 vol 50 mM HEPES (pH 8.0), 360 mM sucrose, 10 PM leupeptin (Sigma, St. Louis, MO), and 1 mM phenylmethylsulfonylfluoride (PMSF; Sigma) at 4 C. The use of a similar homogenization solution during microsome preparation has been shown to inhibit mGHR degradation (22). The homogenate was centrifuged at 10,000 X g for 30 min at 4 C. The

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GHBP

DURING

PREGNANCY

supematant was collected and mixed with 0.5 vol 20 mM HEPES (pH 8.0), 25 mM Car& 10 PM leupeptin, and 1 mM PMSF. This mixture was centrifuged at 10,000 X g for 60 min at 4 C. The pellet was resuspended in 10 ~0120 mM HEPES (pH 8.0), 10 mM CaC12, 10 PM leupeptin, and 1 mM I’MSF and centrifuged at 10,000 X g for 15 min at 4 C. The final pellet was resuspended in 1 ml 20 mM HEPES (pH 8.0)-10 mM MgCl, and frozen at -70 C until treatment with MgCl*. Magnesium chloride treatment of microsomal membrane preparations to remove endogenous GH was performed as described by Kelly et al. (23), with the modifications of Baxter et al. (24). An aliquot of the final preparation was removed for determination of protein concentration (BCA protein assay reagent, Pierce, Rockford, IL), and the remainder was stored frozen at -70 C until assay of GH-binding capacity.

Measurement

of

hepatic microsomal

GH-binding

capacity

Magnesium chloride-stripped hepatic microsomes were diluted in RRA buffer [50 mM HEPES, 10 mM MgClz, 0.1% (wt/vol) RIA grade BSA (Sigma), and 0.01% (wt/vol) Thimerosal (Sigma)] to a volume of 100 ~1. The amount of microsomes added was based on the protein content of the preparation and varied with the physiological state of the animal from which it was obtained. One milligram of microsomal protein was added for microsomes obtained from males, nonpregnant females, and 5-, 7-, and 9-day pregnant mice; 0.75 mg protein was added for microsomes obtained from ll- and 13-day pregnant mice; and 0.5 mg protein was added for microsomes obtained from 15. and 17-day pregnant mice. To test rbGH binding, various concentrations of rbGH plus 25,000 cpm [‘251]iodo-rbGH (SA, 21 &i/fig) diluted in 150 ~1 RRA buffer were added to the microsomes and incubated for 16 h at 22 C. To test mGH binding, various dilutions of mGH plus 25,000 cpm [?I iodo-mGH (SA, 42 &i/pg) diluted in 200 ~1 RRA buffer were added to the microsomes and incubated for 16 h at 22 C. After the 16-h incubation, 0.5 ml ice-cold RRA buffer was added, and the samples were immediately centrifuged at 10,000 X R for 20 min at 4 C. The supernatant was aspirated, and the pellet was assayed for radioactivity in an IsoData 20/20 y-counter. Each concentration of GH was assayed in triplicate. Nonspecific binding was determined by the addition of greater than a 500-fold excess of unlabeled GH.

Measurement

of

serum GH-binding

capacity

The measurement of serum GH-binding capacity was carried out as previously described for human serum (25). Serum was diluted in RRA buffer to a final volume of 200 ~1. Serum dilutions were based on the results of preliminary binding experiments (data not shown) and were chosen to give competitive binding for the hormone concentrations used. Final serum dilutions were 1:90 for samples from male, nonpregnant female, and 5- and 7-day pregnant mice; I:100 for 9-day pregnant mice; 1:250 for ll-day pregnant mice; 1:lOOO for 13-day pregnant mice; and 1:1250 for 15- and 17-day pregnant mice. One hundred microliters of rhGH diluted in RRA buffer were added at increasing concentrations to the diluted serum samples along with 100,000 cpm [‘“Iliodo-rhGH (SA, 149 FCi/wg). Monoclonal antibody 263 (26) (kindly provided by Agen, Australia) was added to a final dilution of 1:500 (vol/vol; 18 fig/ml). The mixture was incubated for 16 h at 4 C and then 2 h at 22 C. One milliliter of 30% (wt/vol) Polvethvlene Glvcol 8000 (Sigma) and 1 ml 0.1% (wt/vol) bovine y-globulin (Sigma) were added; a;d the precipitated GHBP-antibody complexes were centrifuged at 1600 X g for 25 min at 4 C. The supernatants were aspirated, and the pellets were counted. Each concentration of rhGH added was assayed in triplicate. Nonspecific binding for each sample was determined by the addition of greater than a 500-fold excess of rhGH. The affinity of mGHBP for mGH was determined as described previously by our laboratory (27). [‘251]Iodo-mGH (25,000 cpm; SA, 42 &i/pg), radioinert mGH at various concentrations, 15-day pregnant mouse serum [1:250 (vol/vol) final dilution], and a polyclonal antiserum generated against a synthetic peptide corresponding to the mGHBP carboxyl-terminus hydrophilic “tail” (27), as the precipitating antibody (l:l,OOO final dilution added to assay), were mixed in a final volume of 300 ~1 RRA buffer. The mixture was incubated for 16 h at 22 C before 100 ~1 goat antirabbit antiserum, diluted 1:16 (vol/vol) in RRA buffer,

EXPRESSION

878

OF GHR AND

GHBP DURING

PREGNANCY

Endo Voll31.

l

1992 No 2

were added. After incubation for 1 h at 22 C, 100 ~1 30% polyethylene glycol were added, and the tubes were centrifuged at 10,000 X g for 20 min at 4 C. The supematants were aspirated, and the pellets were counted for total radioactivity. Each concentration of mGH added was assayed in triplicate. Nonspecific binding for each sample was determined by the addition of greater than a 500-fold excess of mGH. Concentration GHBP-bound

of total GH in serum and calculation GH and free GH

of

Serum mGH concentrations were determined by RIA, as previously described (28). The concentrations of bound and free GH in individual serum samples were calculated using a computer program described previously (29). The mGH concentration and GH-binding capacity of each individual sample along with the affinity constant of the mGHBP for mGH were used in the cakrlations. The values were calculated using a l-mGH/l-mGHBP model and a l-mGH/2-mGHBP model. Less than a 5% difference in the predicted values of bound US. free mGH was obtained using both models (data not shown). Values calculated from the l-mGH/l-mGHBP model are shown.

eL7 M N'

Scatchurd

analysis of competitive

binding

data

Analysis of competitive binding assays was performed by the method of Scatchard (30). GH binding data analysis and curve fitting were carried out using’ the EBDA program (V2.6) and the Ligand program. Before analvsis of serum GH bindine data, endoeenous serum mGH concentrations assayed by RIA were uled to correct the unlabeled hGH concentration added in each tube. Statistical

analysis

The densitometry data were analyzed by one-way analysis of variance, followed by Fisher’s protected least significant difference test. P < 0.05 was considered significant. The Scatchard data were analyzed for heterogeneity of variance with Bartlett’s test. When significant heterogeneity of variance was present, the data were subjected to log transformation before additional analysis. Nontransformed data are shown in the figures. The data were analyzed by one-way analysis of variance, followed by Scheffe’s F test where appropriate. Pearson product-moment correlations were calculated. P < 0.05 was considered significant.

Results Northern analysis of GHR- and GHBP-encoding liver during pregnancy

messages in

Probing of the Northern blots with the EcoRI/HindIII fragment of clone GHR/BP.23 revealed three specific bands corresponding in size to 1.4, 4.2, and 8.0 kb (Fig. 1, top panel). The 1.4-kb band correspondsto the previously identified 1.2-kb GHBP-encoding mRNA (5). The difference in the size of this messageidentified here and the size previously reported is probably due to differences in RNA standards and gel-running conditions. In addition, the size of the message seems to be more heterogeneous and possibly smaller in the later half of pregnancy (days 9-17 of pregnancy). Due to the differences in total RNA loaded in the lanes (seeFig. 1) and the effects this may have on migration through the gel, it is difficult to determine whether the differences in size of the 1.4-kb messagebetween samples from early and late pregnancy are real or artifactual. For the purposes of this discussion,this messagewill be referred to as the 1.4-kb GHBP-encoding message.The 4.2-kb band correspondsto the previously identified 3.9-kb GHR-encod-

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5

7

9

11 13 1517NPpl7p

FIG. 1. Representative autoradiograph of a Northern blot of liver total RNA. The Northern blot was prepared, as described in Materials and Methods, from RNA samples obtained from animals in the indicated physiological state. Each RNA sample was obtained from a single individual, except for NPp and 17p, which are from pools of RNA from four nonpregnant females or four 17-day pregnant females, respectively. Twenty micrograms of total RNA were loaded in lanes labeled M, NP, 5,7,9, and NPp. Ten micrograms of total RNA were loaded in the lane labeled 11. Five micrograms of total RNA were loaded in lanes labeled 13, 15, 17, and 17~. The Northern blot was probed with the 5’ EcoRI/ Hind111 fragment of the GHR cDNA 23.1 or the ribosomal protein gene L7, as described in Materials and Methods. Exposure time of the film was 20.5 h for the 5’ EcoRI/HindIII GHR cDNA and 27 h for the ribosomal protein gene L7 cDNA. M, Male; NP, nonpregnant female; 5,5 days pregnant; 7, 7 days pregnant; 9,9 days pregnant; 11,ll days pregnant; 13, 13 days pregnant; 15, 15 days pregnant; 17, 17 days pregnant; NPp, nonpregnant female pool; 17p, 17-day pregnant pool.

ing mRNA (5). This messagewill be referred to as the 4.2kb GHR-encoding message.In addition to the previously identified GHBP- and GHR-encoding messagesdiscussed above, an 8.0-kb messagewas also identified in all RNA samplesfrom 9- to 17-day pregnant mice. This RNA will be referred to as the &O-kb GHR/BP RNA, Relative expression during pregnancy

of the 1.4-, 4.2-,

and 8.0-kb

RNA

species

The mean relative expression of the three RNA species identified on Northern blots was calculated from the normalized densitometric data. The upper panel of Fig. 2 shows the mean relative expression of the 1.4-kb GHBP-encoding message.Expression of the 1.4-kb GHBP-encoding message in liver was very low in males and nonpregnant females as well as in animals in the first half of pregnancy. Expression of the 1.4-kb messageincreased steadily during the second half of pregnancy, and by day 17 of pregnancy, relative expression of the 1.4-kb message had increased 20-fold compared with values from nonpregnant mice. The mean relative expression of the 4.2-kb GHR-encoding messagealso increased during pregnancy (Fig. 2, middle panel). The gestational profile of the expression of the 4.2kb message was generally similar to that of the 1.4-kb message,but the magnitude of the increasein the expression

EXPRESSION

OF GHR AND

2.51 l2

1.4

kb

Et3 4.2

kb

PREGNANCY

879

+ 0.93; day 17, 4.14 + 1.06; n = 4; P C 0.05, nonpregnant VS. day 171.When the ratio of the 1.4- to 4.2-kb messages was compared between nonpregnant and late pregnant mice, there was a 2.8-fold increase in the ratio by day 17 of pregnancy. Expression of the 8.0-kb GHR/BP RNA could not be detected in any of the samples from male, nonpregnant female, or 5-, and 7-day pregnant mice with the exposure time of the film used (Fig. 2, bottom panel). There were no significant differences between days of pregnancy when this RNA was detectable; however, there appeared to be a trend toward increasing amounts as pregnancy progressed.

3.0-j

2.0

GHBP DURING

CA T

2.5 2.0 1.5

Liver microsome the mouse

2.0-j 1.54

H

8.0

kb

1 .o 1

A-A&d MNPS

7

9

111315

Pool

Physiological

State

2. Relative expression of the 1.4-, 4.2-, and 8.0-kb GHR/GHBP RNA species in liver. Northern blots were prepared and probed, and xray film was exposed, as described in Fig. 1. Densitometric analysis was carried out, as described in Materials and Methods. Each bar shows the mean + SE of the relative expression of the different GHR/GHBP RNA species from four separate Northern blots, except for males, which are the means from three Northern blots. Each separate Northern blot contained RNA samples from different individuals, except NPp and 17p, which were used as internal controls, as described in Materials and Methods. The layout of the Northern blots was identical to that in Fig. 1. Bars within a given panel containing the same letter are not statistically different from each other (P 5 0.05). The absence of a bar indicates that the message was not detectable. Abbreviations are the same as in Fig. 1. FIG.

of the 4.2-kb messagebetween nonpregnant and late pregnant mice was lower than that of the 1.4-kb message,with an 8-fold difference in expression of the 4.2-kb message between nonpregnant and 17-day pregnant animals. The ratio between the 1.4-kb GHBP-encoding and the 4.2kb GHR-encoding messageswas calculated from the densitometry data from each autoradiograph, and ratio means were calculated for each physiological state. During pregnancy there was a general trend toward a higher ratio of 1.4kb GHBP-encoding messageto 4.2-kb GHR-encoding message [mean f SE ratio of 1.4- to 4.2-kb message(ratio of relative OD units): nonpregnant females, 1.49 f 0.20; day 5 of pregnancy, 2.49 f 0.41; day 7, 1.43 If: 0.24; day 9, 2.93 f 0.33; day 11, 2.58 + 0.30; day 13, 2.53 f 0.93; day 15, 3.46

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GH-binding

capacities during pregnancy

in

The GH-binding capacity of liver from pregnant female, nonpregnant female, and male mice is shown in Fig. 3. The GH-binding capacity of microsomes from 15-day pregnant mice was 16-fold greater than the GH-binding capacity of microsomesfrom nonpregnant female mice. There were no significant differences in the calculated affinity constants between any of the physiological states(data not shown). To validate the use of rbGH for estimation of the GHbinding capacities of liver microsomesand also to determine the affinity constant for mGH, we tested mGH binding to liver microsomes from nonpregnant and 15-day pregnant mice. Five samplesfrom each group were used to generate binding data, which were plotted by the method of Scatchard (30). No significant differences in the mean affinity constants between nonpregnant and 15-day pregnant mice were observed when either mGH or rbGH were used (data not shown). There were no significant differences in the mean GH-binding capacity estimateswhen either mGH or rbGH s E L 0

400

TC

a ”

MALENP

a

a

D5

D7

D9

DllD13D15D17

3. GH-binding capacities of liver microsomes during pregnancy, determined by Scatchard analysis. Liver microsome preparation, [iz51] iodo-rbGH-binding. and Scatchard analvsis were as described in Muterials and Method;. Each bar shows the-mean + SE of data from three Scatchard plots. Each Scatchard plot was generated from binding data derived from microsomes prepared from a pool of two individual livers from mice in the indicated physiological state. The GH-binding capacity is in femtomoles per mg microsomal protein. Bars that do not share the same letter above them are statistically different from each other (P < 0.05). Male, Male; NP, nonpregnant female; D5,5 days pregnant; D7,7 days pregnant; D9,9 days pregnant; Dll, 11 days pregnant; D13, 13 days pregnant; D15,15 days pregnant; D17,17 days pregnant. FIG.

EXPRESSION OF GHR AND GHBP DURING PREGNANCY

880

Endo. Vol131.

1992 No 2

was used (data not shown). The mean affinity constant estimated by Scatchard analysis of the mGHR for mGH was 1.7 X 10” + 0.3 X 10” M-’ (n = 10). Serum GH-binding

capacities during pregnancy

in the mouse

Figure 4 shows the mean serum GH-binding capacities of serum from pregnant female, nonpregnant female, and male mice. The GH-binding capacity was low in males and nonpregnant females. In pregnant animals, the GH-binding capacity increasedsignificantly at midpregnancy and remained high until the end of pregnancy. Comparison of the mean serum GH-binding capacity of nonpregnant or early pregnant femaleswith the mean serum GH-binding capacitiesof late pregnant femalesindicates that there is a 30- to 50-fold increasein serum GHBP levels between early pregnant and late pregnant mice. There were no significant differences in the mean affinity constants between the groups (data not shown). The mean affinity constant of the mGHBP for mGH was predicted by Scatchard analysis to be 8.3 X 10’ + 1.6 x 10’ M-’ (n = 4). The predicted value of the mGHBP affinity constant is statistically different from the predicted value of the mGHR affinity constant (P < 0.05).

Predicted

-

GHBP-bound

and free mGH during

The total mGH concentration was measuredin the serum samplesin which GH-binding capacity was determined. The mean serum total mGH concentration of nonpregnant mice and mice through day 11 of pregnancy was lessthan 1.OnM (Fig. 5, top). Between days 13 and 17 of pregnancy there was a steady increasein the concentration of total mGH in serum, with values on day 17 of pregnancy being about 9-fold greater than those in nonpregnant mice. After parturition, the concentration of total mGH declined rapidly. In nonpregnant animals and most animals between days 5 and 11 of pregnancy, the predicted concentration of GHBPbound mGH was below 0.3 nM (Fig. 5, bottom). On day 13 of pregnancy, the concentration of GHBP-bound mGH be-

Bound

l

Free

[GHI IGHI

w NP

Total mGH and predicted pregnancy

q

D5

D7

DV

Dll

D13

D15

D17

Ll

Day of Pregnancy FIG. 5. Total ing pregnancy. concentrations Serum samples same as those three samples. abbreviations

and predicted free and bound mGH concentrations durCalculation of the predicted free and bound mGH was determined as described in Materials and Methods. for NP, D5, D7, D9, Dll, D13, D15, and D17 were the used in Fig. 4. Values are the mean + SE of values from Ll, Day 1 of lactation (19 days after vaginal plug). Other are explained in Fig. 3.

gan to rise and then increased steadily until day 17. On day 1 of lactation the concentration of GHBP-bound mGH fell. The predicted free mGH concentration changed very little between nonpregnant and pregnant animals (Fig. 5, bottom) and appeared to be tightly regulated in a very narrow range below 0.5 nM. Discussion

g V * .Z

ii Q 0 F z

400 300 -

200 loo-

? 3-

-c

.a

a

a

o-

a *

MALENP

D5

D7

D9

DllD13D15D17

FIG. 4. Serum GH-binding capacities during pregnancy. The serum GHBP concentration was determined by Scatchard analysis. Each bar shows the mean + SE of three or four Scatchard plots. Each Scatchard plot was generated from binding data from the serum of a single mouse at the indicated physiological state. Statistical analysis and abbreviations are described in Fig. 3.

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The factors regulating the increase in the GHR- and GHBP-encoding messagesduring pregnancy are incompletely understood. There is a large increase in the serum GH concentration in the second half of pregnancy in the mouse. Mice hypophysectomized on day 11 of pregnancy failed to demonstrate the increase in both the GHR- and GHBP-encoding messagesseen on day 14 of pregnancy in control mice, and replacement with GH partially restored the GHR and GHBP messagelevels to control values (12). Interestingly, in the latter study, a difference in the magnitude of the increase between GHR- and GHBP-encoding messages was present, with the greater effect being on the GHBPencoding message.Together thesedata suggesta role for GH in the differential regulation of steady state levels of GHBPand GHR-encoding messagesduring pregnancy. However, other factors undoubtedly also play a role. Maternal serum GH concentration begins to increaseon day 13 of pregnancy, which is after the increase in GHR and GHBP mRNAs, and

EXPRESSION

OF GHR AND GHBP

therefore, GH is probably not the factor involved in the initial induction of GHR/BP mRNA observed on day 9. Other endocrine changesthat might be responsible for the regulation of steady state levels of GHR and GHBP messagesoccur during midpregnancy in the mouse. Serum placental lactogen-I (31) and androgen (32) concentrations rise rapidly beginning on about day 8 of pregnancy. At the same time that these increasesare occurring, pituitary PRL concentrations decreasesharply (6). Some of these endocrine changes may be involved, directly or indirectly, in the regulation of GHR and GHBP mRNA steady state levels. We suspectthat the 8.0-kb messageidentified in this study is an unspliced precursor to the 1.4- and/or 4.2-kb GHR/BP messages,A 10.5-kb RNA specieshas been identified for the PRL receptor in rabbit mammary gland (33). This RNA was shown to be a primary transcript of nuclear origin. Identification of the exact nature of the 8.0-kb RNA speciescould provide valuable insight into the generation of the 1.4- and 4.2-kb messages. The timing of the increasein hepatic GH-binding capacity parallels the increase in steady state liver GHR-encoding mRNA. However, the magnitude of the hepatic GH-binding capacity increase is roughly 2-fold greater than that of the increase in liver steady state mRNA. The increase in serum GH-binding capacity also parallels the increase in steady state liver GHBP-encoding mRNA, and the magnitude of the increasein GH-binding capacity is roughly twice that of the increase in steady state mRNA. The 2-fold difference in the increases of RNA and proteins may reflect differences in hepatic RNA translation efficiencies, protein stablities, and/ or clearanceduring the second half of pregnancy. The affinity of the hepatic GH-binding site is similar to that of the high affinity GH-binding site recently demonstrated in hepatic microsomesof late pregnant humans (11). However, in hepatic microsomesfrom nonpregnant and late pregnant women, there is also a low affinity GH-binding site. High and low affinity GH-binding sites have been identified in the steeraswell (34). Increasesduring pregnancy in hepatic GH-binding capacities have been observed in humans (ll), rabbits (35), and rats (lo), demonstrating that an increasein hepatic GH-binding capacity during pregnancy is common to a number of widely divergent species;however, the magnitude of this increase seemsto vary considerably between species. The increase in GH-binding capacity in maternal serum during pregnancy is consistent with previous reports from this laboratory where GH-binding increasewas measuredby relative binding (8) or the concentration of mGHBP was measured by RIA (27). A recent report suggeststhat the GHBP may be regulated differently in the rat and mouse (10). In the rat the serum GH-binding capacity increases approximately 2-fold during pregnancy compared with the 30- to 50-fold increasein the mouse. The significance of this difference between rats and mice is unclear. No data exist from other specieson the regulation of serum GH-binding activity during pregnancy. The affinity of the hepatic GHR was 20-fold greater than the affinity of the GHBP, using mGH as the ligand. The

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DURING

PREGNANCY

881

binding data used to generate the Scatchard plots for determining the affinity constants of both the GHR and GHBP were produced with the same batch of [‘251]iodo-mGH as radioligand, the samebatch of radioinert mGH as competing hormone, and the samebuffer preparation. The assayswere conducted within 48 h of each other, each with multiple samples. However, significant differences do exist between the methodologies used in the two assaysin the separation of bound radioactive hormone. The assay of GHBP uses a polyclonal antiserum to immunoprecipitate the GHBP out of solution. Theoretically, the use of antiserum against the hydrophilic “tail” of the GHBP to immunoprecipitate the GHBP could affect the affinity of the GHBP for GH (36). The recent determination of the crystal structure of a complex between human GH and the soluble hormone-binding domain of the human GHR (residues l-238) has demonstrated that the the carboxyl-terminus of this molecule is not involved in hormone binding (37). The antiserum we used in this study is to a region in the mGHBP that would be extended beyond the carboxyl-terminus of the human GHRbinding protein and should not affect the affinity constant of the GHBP. The factors underlying the differences in affinity between the mGHR and mGHBP are not known. The GH-binding sitesof the mGHBP and mGHR are identical in amino acid sequence(5), but may differ in glycosylation patterns. Differences in affinities may result from differences associated with the GHR being membrane-bound and the GHBP being soluble. Interaction of the GHR with other membrane-associatedproteins may also contribute to the differences in affinity between the GHR and GHBP. The amount of free GH in the serum appears to be very tightly regulated below 0.5 nM, despite increasesin total GH concentration in late pregnancy of greater than 4.0 nM. At the sametime, the amount of GHBP with bound GH is less than 10% of the total GHBP available. These results demonstrate that there is a large excessof free GHBP relative to the amount of total GH in the late pregnant mouse and suggest that the GHBP may act as a reservoir for GH increases.Dynamic equilibrium would allow a small fraction of the GH to be in a free pool. If releaseoccurred near a GHbinding site on the liver or other tissueswith GHR, the GHR, due to its higher affinity for GH, would tend to bind the released GH. It is possible that during pregnancy, as a consequenceof the elevated GHBP concentration, only tissueswith elevated GHR would retain maximum responsivenessto GH. The exact functions of the GHBP are not known. Experimental data using homologous GH and glycosylated GHBP are not available. The data presentedhere are consistent with an important role for GHBP in regulating availability of GH to target tissuesduring pregnancy. Alternatively, the GHBP may be involved in GH bioactivity by interacting directly at target tissues. Acknowledgments We thank Dr. Judith Campisi for providing ribosomal protein gene L7, and Dr. Jonathan figure preparation.

us with the cDNA for the Southard for his help in

882

EXPRESSION

OF GHR

AND

GHBP

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