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Endocrinology Vol. 143, No. 11 4310-4319
Copyright © 2002 by The Endocrine Society

ARTICLE

Growth Hormone, Acting in Part through the Insulin-Like Growth Factor Axis, Rescues Developmental, But Not Metabolic, Activity in the Mammary Gland of Mice Expressing a Single Allele of the Prolactin Receptor

Gordon J. Allan, Elizabeth Tonner, Michael C. Barber, Maureen T. Travers, John H. Shand, Richard G. Vernon, Paul A. Kelly, Nadine Binart1 and David J. Flint1

Institut National de la Santé et de la Recherche Médicale, Unité 344, Faculté de Médecine Necker (P.A.K., N.B., D.J.F.), 75730 Paris, France; and Hannah Research Institute (G.J.A., E.T., M.C.B., M.T.T., J.H.S., R.G.V., D.J.F.), Ayr KA6 5HL, United Kingdom

Address all correspondence and requests for reprints to: Dr. D. J. Flint, Hannah Research Institute, Ayr KA6 5HL, United Kingdom. E-mail: flintd{at}hri.sari.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The heterozygous prolactin (PRL) receptor (PRLR+/-) mouse fails to develop a fully functional mammary gland at the end of the first pregnancy and shows markedly impaired lobuloalveolar development and milk secretion in young females. PRL and GH, acting through the IGF system, have interactive effects to enhance epithelial cell survival. Thus, we propose that a reduction in the expression of the PRLR may lead to increased IGFBP-5 expression (proapoptotic) and that GH may rescue mammary development by increasing IGF-I, an important mitogen and survival factor for the mammary epithelium. Mammary IGF-binding protein-5 (IGFBP-5) concentrations and plasmin activity in PRLR+/- mice were increased on d 2 postpartum, indicative of increased cell death and extracellular matrix remodeling. After GH treatment, a restoration of mammary alveolar development and a reduction in the activities of IGFBP-5 and plasmin were observed. Despite the severely impaired mammary development in PRLR+/- mice, both mRNA and protein expression for caseins and acetyl-coenzyme A (acetyl-CoA) carboxylase and acetyl-CoA caboxylase-{alpha} mRNA increased at parturition, although not to the extent in wild-type animals. Surprisingly, GH treatment actually led to a further decrease in milk protein and acetyl-CoA carboxylase-{alpha}expression when expressed per cell. This was confirmed by the smaller alveolar size, the relative paucity of milk in the mammary glands of GH-treated animals, and the inability of their pups to gain weight. In a subsequent study IGFBP-5 was administered to wild-type mice and produced a 45% decrease in mammary DNA content, a 30% decrease in parenchymal tissue, and impaired lactation. These results suggest that GH can improve mammary development in PRLR+/- mice, but that it fails to enhance metabolic activity. This may be due to the maintenance by GH/IGF-I of a proliferative, rather than a differentiative, phenotype.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROLACTIN (PRL) PLAYS a key role in mammary development and milk synthesis in the mouse (1, 2, 3). To examine its role in various developmental processes we developed a PRL receptor knockout (PRLR ko) mouse. This involved a germline null mutation of the PRLR produced by replacing exon 5 with the thymidine kinase-NEO cassette (4). Normal mammary development occurred up to puberty, but subsequent ductal branching was impaired in the PRLR ko mice, where terminal end buds persisted at the ductal ends. Alveolar development was absent, and milk synthesis failed to occur. Homozygous PRLR ko females are sterile and exhibit altered maternal behavior and bone development (5, 6, 7). At least part of the reproductive phenotype is due to progesterone deficiency, as progesterone treatment rescued development of preimplantatory eggs and embryo implantation (8). Progesterone also increased ductal side-branching in the mammary glands of PRLR ko mice. Surprisingly, 8-wk-old heterozygous (PRLR+/-) females, although displaying normal fertility, also showed impaired mammary development during pregnancy and an inability to support their offspring (5).

Mammary development is dependent upon IGF-I and -II. Both have been shown to be mitogenic and to act as survival factors for the mammary epithelium in transgenic mice (9, 10, 11, 12, 13, 14). The effects of the IGFs are modulated by a family of six IGF-binding proteins (IGFBPs), and we have proposed that one of these, IGFBP-5, which is expressed during mammary involution, serves to induce apoptotic cell death in the mammary gland by blocking the actions of IGF-I (15, 16, 17). We have also shown that PRL is the major factor responsible for inhibiting the synthesis of IGFBP-5 by the mammary epithelium (17), and by inference, we proposed that this is part of the mechanism by which PRL interacts with GH to promote cell survival. GH increases IGF-I synthesis, whereas PRL inhibits IGFBP-5 synthesis, to maximize the effects of IGF-I (17).

Several studies have argued that GH, rather than PRL, has a major role in rodent mammary gland development (as distinct from milk synthesis) (18, 19), acting at least in part by stimulating circulating concentrations of IGF-I (20, 21, 22). Recently, we showed that GH stimulates STAT5 (signal transducer and activator of transcription-5) preferentially, though not exclusively, in the mammary stroma, supporting an indirect action on the mammary gland, but also demonstrating a potential direct lactogenic ability in PRLR-null mammary epithelium (23). Expression of GH receptors in both stromal and epithelial components of the mouse mammary gland have also been described (24).

We have previously demonstrated that PRL and GH play supporting roles in various aspects of mammary gland function, including established milk synthesis (25), casein synthesis (26), lipid synthesis (27, 28), and mammary cholesterol metabolism (29), although the role of PRL was always quantitatively dominant. Subsequently, we offered a mechanistic explanation for this interaction when we described their interactive roles in maintaining epithelial cell survival (27, 30, 31) and controlling mammary plasminogen activation to plasmin. Plasminogen activation occurs when cellular function or alveolar integrity is compromised in the mammary gland and results in the activation of extracellular matrix degradation (32, 33, 34). In our model, GH and PRL act to optimize mammary epithelial cell survival in a process involving suppression of IGFBP-5 expression by PRL (17) and suppression of plasminogen activation by both GH and PRL (34). This enhances the survival effects of GH, acting through stimulation of IGF-I secretion. We therefore hypothesize that if PRL actions were impaired in the PRLR+/- mouse, IGFBP-5 synthesis and plasmin production might be increased in the mammary epithelium, leading to enhanced rates of cell death and extracellular matrix degradation, which would result in impaired mammary gland function. If this were the case, GH treatment, possibly acting through increased IGF-I synthesis, might redress this imbalance, leading to improved epithelial cell survival and normal mammary development. Furthermore, we propose that administration of exogenous IGFBP-5 to wild-type animals would result in impaired mammary gland development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All experimental designs and procedures were in agreement with the guidelines of the animal ethics committee of the French Ministère de l’Agriculture. PRLR+/- mice and their wild-type siblings (+/+), approximately 8 wk old, were in the inbred 129/Sv background and were mated with 129/Sv male mice. The day of a vaginal plug was designated d 1 of pregnancy. Mice were housed on a 12-h light, 12-h dark cycle at 22 C with ad libitum access to food and water. On d 14 of pregnancy, three +/- and three +/+ mice were killed by cervical dislocation. Six of the PRLR+/- animals were treated from d 14–21 of pregnancy with recombinant bovine GH, administered sc at 300 µg/d in 20% polyvinylpyrrolidone (wt/vol; in 0.15 M NaCl) as a delay vehicle. Six PRLR+/- mice served as controls and received vehicle only. Wild-type animals of equivalent age were mated and remained untreated throughout. At parturition, pups were weighed, then were weighed again 24 h later and examined for the presence of milk in their stomachs.

All dams were killed by cervical dislocation, and blood was collected by cardiac puncture for determination of serum IGF-I concentrations (25). Where indicated, the fourth right abdominal gland was used to prepare a whole mount of the mammary gland (35), and the second, third, and fourth left glands were frozen in liquid nitrogen and stored at –80 C for subsequent analyses as described below.

Determination of casein and whey acid protein (WAP) concentrations in mammary tissue
Mammary tissue (30 mg/ml wet weight) was homogenized at 13,000 rpm for 15 sec using a Polytron homogenizer (Kinematica, Luzern, Switzerland). The protein concentration was determined by the method of Bradford (36). Five micrograms of membrane protein along with mouse {alpha}- and ß-casein and WAP standards were resolved by SDS-PAGE and blotted onto nitrocellulose before probing with rabbit antiserum to mouse caseins (1:2,000) or mouse WAP (1:10,000), and detection was performed using antirabbit alkaline phosphatase conjugate (1:10,000) (37). Bands were quantified by densitometric analysis using a Molecular Dynamics, Inc., PhosphorImager 445SI and were analyzed with ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA).

Ribonuclease protection assays
A 438-nucleotide fragment of mouse acetyl-coenzyme A (acetyl-CoA) carboxylase-{alpha} [ACC-{alpha}; corresponding to nucleotides 3854–4292 of the ovine sequence (38)] was generated by PCR of adipose tissue cDNA with primers (5'-GTAGAATTCCAGTTCATGCTGCCCACATC-3' and 5'-GCCGGATCCCATGCTCACCAGAGTAGCT-3'). The ACC-{alpha} cDNA was cloned into pGEM-7zf+ and used to generate a 32P-labeled 483 nucleotide antisense transcript (39). A mouse ß-casein cDNA (40) was prepared by PCR of lactating mammary gland cDNA with primers (5'-TATGAATTCATGAAGGTCTTCATCCTCGCCTGCTT-3' and 5'-TATGAATTCTTCAGGAGAAATGACAGGCCCAAGAGA-3'). The resulting 327-bp fragment was cloned into pGEM-7zf+ and used to generate 32P-labeled antisense transcript as described above. Tissue samples were powdered in a mortar and pestle using liquid nitrogen and homogenized in 5 M guanidinium isothiocyanate and 100 mM EDTA, pH 7.0, using a constant tissue to volume ratio. Aliquots (40 µl) of these were hybridized to the antisense cRNA and subsequently digested using ribonuclease A/T1 and proteinase K as described by Firestein et al. (41). In addition, standard amounts of sense transcript were hybridized to the antisense transcript as positive controls. After extraction with phenol and chloroform, the samples were precipitated twice and rinsed with 80% (vol/vol) alcohol. Samples were dried, resuspended, and, after denaturing in formamide loading buffer at 85 C, resolved on a 6% (wt/vol) acrylamide/7 M urea sequencing gel (42). After drying, the gels were exposed to a Kodak phosphor screen overnight (Rochester, NY). The resulting images were scanned using a PhosphorImager 445 SI (Molecular Dynamics, Inc.), and the density of individual bands was determined using ImageQuant software (Molecular Dynamics, Inc.).

DNA content
The DNA content of the homogenates was measured by a modification of the method of Labarca and Paigen (43). In this, tissue samples were homogenized in 5 M guanidinium isothiocyanate and 100 mM EDTA, pH 7.0, and extracted with 4 vol water-saturated chloroform before being assayed as described.

Acetyl-CoA carboxylase
Total acetyl-CoA carboxylase activity (activity after preincubation with 20 mM citrate to activate any enzyme in the inactive state) was determined as described previously (27).

Determination of mammary IGFBP-5 expression
Twenty micrograms of mammary homogenates were subjected to electrophoresis, Western blotting, and probing with 125I-labeled IGF-I as described previously (17).

Plasmin/plasminogen activity
These were determined in mammary homogenates as described previously (34) involving the measurement of plasmin (minus urokinase) and plasminogen (plus urokinase) using the substrate Val-Leu-Lys-p-nitroanilide hydrochloride.

Histological analysis
Mammary samples used for whole mount studies were subsequently embedded in paraffin, and 5- to 7-µm sections were cut and stained with hematoxylin and eosin before microscopic analysis and photography.

Production of recombinant rat IGFBP-5
Rat IGFBP-5 cDNA, without the signal peptide-encoding sequence, was cloned into the pGEX 6P-1 vector (Amersham Pharmacia Biotech, Arlington Heights, IL) between BamHI and EcoRI in the multiple cloning site. Fifty nanograms of this construct were then used to transform the Origami B (DE3) pLysS strain of Escherichia coli, and the cells were incubated overnight at 37 C in 10 ml Luria-Bertoni medium containing 125 µg/ml ampicillin and 30 µg/ml chloramphenicol. After a 40-fold dilution into fresh Luria-Bertoni/ampicillin/chloramphenicol, the cells were regrown to midlog phase (E600nm = ~0.6), then the expression of IGFBP-5, as a glutathione-S-transferase fusion protein, was induced by addition of 1 mM isopropyl ß-D-thiogalactoside and allowed to proceed at 25 C overnight. Cells were harvested by centrifugation at 1,500 xg for 15 min, washed once in 50 ml PBS, and resuspended in 10 ml PBS containing protease inhibitors (Roche, Indianapolis, IN). The suspension was frozen and thawed once to lyse the cells, then the bacterial DNA was sheared by three 30-sec cycles of sonication (KT-40, Kontes Co., Vineland, NJ; 4-mm probe, full power) with cooling on ice. Insoluble material was removed by centrifugation at 11,000 x g for 30 min, then the supernatant was filtered through a 0.45-µm pore size membrane and incubated overnight at 4 C with 1 ml (packed volume) washed glutathione-Sepharose (Amersham Pharmacia Biotech). The suspension was decanted into a disposable plastic column (Bio-Rad Laboratories, Inc., Hercules, CA), and unbound material allowed to flow through, then the glutathione-Sepharose was washed twice with 10 ml PBS and once with 10 ml cleavage buffer [50 mM Tris (pH 7.0), 150 mM NaCl, and 1 mM EDTA]. The column was sealed, and the glutathione-Sepharose was resuspended in 2 ml cleavage buffer containing 160 U PreScission protease (Amersham Pharmacia Biotech) (44, 45). After 4 h at room temperature with hourly resuspension, the column was reopened, and the cleaved IGFBP-5 was recovered in the eluate. Glutathione-S-transferase and PreScission protease remained bound to the glutathione-Sepharose. IGFBP-5 remaining in the column was recovered by washing with 10 ml cleavage buffer. A 400-ml bacterial culture typically yielded about 2 mg IGFBP-5.

Effects of rIGFBP-5 on mammary function
Wild-type mice were injected sc with 50 µg recombinant IGFBP-5 dissolved in 50 mM Tris HCl, pH 7.0, containing 150 mM NaCl and 1 mM EDTA twice daily for 7 d, beginning on d 16 of pregnancy. Control mice received excipient only. Litters were adjusted to eight pups, and daily litter weight gain was determined until the dams were killed on d 5 of lactation (18 h after the final injection of IGFBP-5). Mammary glands were removed, weighed, and stored at -80 C for DNA determination or were fixed in 4% formaldehyde, sectioned, and stained with hematoxylin and eosin for determination of parenchymal areas using the LUCIA image analysis system (Nikon, France SA, Champigny sur Marne).

Statistical analysis
Statistical comparisons were performed on log-transformed data using ANOVA, followed by post hoc multiple comparisons using the Bonferroni correction.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PRLR+/- mice, mated at approximately 8 wk of age, showed impaired mammary development during pregnancy. Whole mounts (Fig. 1Go) and histological sections (Fig. 2Go) showed that wild-type animals had mammary glands in which the mammary alveoli filled the majority of the fat pad (Figs. 1dGo and 2dGo), whereas in the PRLR+/- animals alveolar development was significantly reduced (Figs. 1bGo and 2Go, a and b), appearing more comparable to d 14 of pregnancy (Fig. 1aGo). Figure 2Go, a and b, represent the extremes in PRLR+/- mice, with one animal showing abnormal, essentially ductal structures (Fig. 2aGo), and the other showing some alveolar development (Fig. 2bGo). GH treatment for the last 7 d of pregnancy was able to overcome this defect, leading to essentially normal mammary development in which the majority of the fat pad was filled with alveolar structures (Figs. 1cGo and 2cGo). However, alveolar size appeared to be smaller than that in wild-type animals (compare Fig. 2Go, c and d), suggesting that filling of alveoli with milk was less apparent in the GH-treated animals. Consistent with the developmental picture, IGFBP-5 and plasmin concentrations were increased 3- to 4-fold in PRLR+/- mammary tissue (Fig. 3Go). These increases were completely prevented by GH treatment. Serum IGF-I concentrations on d 2 of lactation were unaffected in animals expressing one allele of the PRL receptor (mean ± SEM, 27.3 ± 3.2 nmol/liter, compared with 28.8 ± 3.7 nmol/liter in wild-type animals). In contrast, they were significantly elevated in animals receiving GH (85.5 ± 12.3 nmol/liter; P < 0.01 compared with wild-type animals). The histological results were also confirmed, quantitatively, by determining mammary weight and DNA content (DNAt) (Fig. 4Go). These parameters increased 2- and 4-fold, respectively in wild-type animals, whereas neither increased in PRLR+/- mice, remaining similar to those on d 14 of pregnancy. GH treatment resulted in mammary weights that were similar to those in wild-type mice and actually showed an increased DNA content compared with wild-type mice. These results demonstrated that GH could, in a qualitative sense, normalize mammary gland development. Milk could also be detected in the stomachs of the pups of all GH-treated +/- animals, although it was not as abundant as in pups of wild-type animals. This suggested that milk synthesis was not stimulated to the same extent in GH-treated PRLR+/- mice as in wild-type mice. Further evidence for this was obtained from pup weight gains, which were 0.32 ± 0.07 (mean ± SEM), -0.01 ± 0.06, and -0.03 ± 0.06 g in wild-type, PRLR+/-, and PRLR+/- mice given GH, respectively, indicating an inability of GH to support pup weight gain during the first 24 h postpartum. We therefore examined various metabolic aspects of the mammary gland in greater detail.



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  		Figure 1. Whole mount analysis of mammary glands on d 14 of pregnancy (a) or d 2 of lactation (b) in PRLR+/- mice, a PRLR+/- mouse receiving GH on d 14–21 of pregnancy (c), or a wild-type (+/+) mouse (d). Note the sparcity of mammary ductal invasion and limited alveolar development in b compared with c and d.

 


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  		Figure 2. Hematoxylin and eosin staining of mammary gland sections on d 2 of lactation from two different PRLR+/- mice (a and b), PRLR+/- mice receiving GH on d 14–21 of pregnancy (c), or wild-type (+/+) mice (d). Note that alveolar tissue has completely filled the fat pad in c and d, whereas in a, large amounts of adipose tissue are evident, and mammary tissue is largely ductal, with relatively small numbers of alveoli. b, One PRLR+/- mouse that showed some alveolar development, but even here alveoli filled less than 50% of the mammary fat pad. Also note the enlarged alveolar volume, indicating greater milk accumulation in d compared with b and c. A, Adipocytes; M, mammary alveolar tissue;D, duct.

 


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  		Figure 3. IGFBP-5 ({blacksquare}) and plasmin ({square}) activity in mammary tissue from wild-type and PRLR+/- mice on d 2 of lactation. Values are the mean ± SEM of six mice, except for wild-type animals, where n = 3 for IGFBP-5 measurements. *, P < 0.05 compared with wild-type or PRLR+/- mice receiving GH.

 


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  		Figure 4. Mammary gland weight ({square}) and total DNA content ({blacksquare}) on d 14 of pregnancy or on d 2 of lactation in wild-type, PRLR+/-, or PRLR+/- mice receiving GH on d 14–21 of pregnancy. Values are the mean ± SEM of five to nine mice. **, P < 0.01; ***, P < 0.001 (compared with d 14 of pregnancy and PRLR+/-).

 
When expressed per cell, ß-casein mRNA increased 17-fold between d 14 of pregnancy and d 2 of lactation in wild-type animals (Fig. 5AGo) and 16-fold in PRLR+/- mice, but only 7-fold in PRLR+/- mice given GH. ß-Casein protein was below the detection limit on d 14 of pregnancy, but increased dramatically in wild-type animals on d 2 of lactation (Fig. 5BGo). Casein content per cell in PRLR+/- was, however, only approximately 25% of that in wild-type animals, suggesting less efficient translation of mRNA into protein. Casein content per cell was also markedly decreased in PRLR+/- mice given GH (Fig. 5BGo). Similar findings were evident for {alpha}-casein and WAP, where both {alpha}-casein and WAP in PRLR+/- mice given GH were only 15% of wild-type values (results not shown). The effects on ACC-{alpha} mRNA were similar (Fig. 6AGo). ACC-{alpha} mRNA increased 5-fold in wild-type animals compared with d 14 of pregnancy, 4-fold in PRLR+/- mice, but only 2.5-fold in PRLR+/- mice given GH. The effect on acetyl-CoA carboxylase enzyme activity was even more dramatic. Enzyme activity increased 9-fold between d 14 of pregnancy and d 2 of lactation in wild-type animals (Fig. 6BGo), increased 2.5-fold in PRLR+/- mice, and actually decreased by 50% in PRLR+/- mice given GH compared with d 14 of pregnancy.



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  		Figure 5. Steady state mRNA levels (A) and protein concentration (B) for ß-casein expressed per unit of DNA on d 14 of pregnancy ({square}) or on d 2 of lactation in wild-type (light-gray bars), PRLR+/- (mid-gray bars), or PRLR+/- mice receiving GH on d 14–21 of pregnancy ({blacksquare}). Values are the mean ± SEM of three to nine mice. Values with different superscripts differ: a vs. b or c, P < 0.001; b vs. c, P < 0.05. *, P < 0.05 compared with PRLR+/- given GH. UD, Below detection limit.

 


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  		Figure 6. Steady state ACC-{alpha} mRNA levels (A) and total activity (B) of acetyl-CoA carboxylase expressed per unit DNA on d 14 of pregnancy ({square}) or on d 2 of lactation in wild-type (light-gray bars) PRLR+/- (mid-gray bars) or PRLR+/- mice receiving GH on d 14–21 of pregnancy ({blacksquare}). Values are the mean ± SEM of four to seven mice. *, P < 0.05 compared with d 14 of pregnancy and PRLR+/- given GH.

 
When expressed per total mammary gland, the picture was somewhat different, as all parameters were markedly reduced in PRLR+/- mice (Table 1Go) as a result of their much lower cellular content. By contrast, GH treatment of PRLR+/- mice increased total ß-casein mRNA such that it attained approximately 50% of wild-type values, although ß-casein protein was less than 30% of wild-type values. Most striking was the effect on acetyl-CoA carboxylase in PRLR+/- mice given GH, where ACC-{alpha} mRNA was 75% of wild-type values, but expressed enzyme activity was only 5% of wild-type values, indicating dramatically impaired enzyme activation and, as a consequence, reduced fatty acid synthesis (Table 1Go). Hybridization of the 483-nucleotide cRNA probe to lactating mammary gland RNA produced protected fragments of 438 and 382 nucleotides (results not shown). These correspond to ACC-{alpha} mRNAs either containing (438-nucleotide species, long transcript) or lacking (382-nucleotide species, short transcript) a 24-nucleotide exonic sequence. The ratio of short to long forms of ACC-{alpha} mRNA increased from 1.8 ± 0.4 (mean ± SEM) on d 14 of pregnancy to 5.4 ± 0.3 on d 2 of lactation in wild-type mice. It also increased in PRLR+/- mice (4.0 ±0.68) and PRLR+/- mice given GH (4.6 ±1.2). Thus, the lack of a response in acetyl-CoA carboxylase activity after GH treatment occurred despite both an increase in total ACC-{alpha}mRNA concentrations and a switch in short/long form mRNA ratios, which is characteristic of the onset of lactogenesis.


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  		Table 1. Protein and steady state mRNA levels for ß-casein, {alpha}-casein, WAP, and activity of acetyl-coenzyme A carboxylase in mammary glands of PRL receptor +/- mice

 
To determine a causal relationship between increased IGFBP-5 expression and impaired mammary development and function, we treated wild-type females with recombinant IGFBP-5 during the periparturient period. This resulted in a significant decrease of about 40% in litter weight gain, an index of milk secretion (Fig. 7AGo), and decreases in mammary gland weight (Fig. 7BGo), DNA content (Fig. 7CGo), and the proportion of parenchymal tissue (Fig. 7DGo). Histological analysis revealed large areas of adipose tissue remaining in the IGFBP-5 animals (highlighted in Fig. 8Go) compared with the typical mammary development in control animals, where adipose tissue is present in much smaller amounts. These results indicate that IGFBP-5 is able to inhibit both mammary gland development and function.



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  		Figure 7. Litter weight gain (A; squares, control mice; circles, IGFBP-5 treated), mammary weight (B), total DNA (C), and proportion of parenchymal tissue (D) in control mice () or mice treated for 7 d with rIGFBP-5 ({blacksquare}). Values are the mean and SEM for five to seven mice per group. *, P < 0.05; **, P < 0.01 (compared with control animals).

 


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  		Figure 8. Histological sections of two control animals (a and b), showing the typical alveolar epithelium, and of two animals treated with IGFBP-5 (c and d), showing sections of the gland where mammary epithelium is much less extensive and where large areas of adipose tissue remain.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
GH has previously been shown to stimulate mammary growth in rodents (18), at least in part by stimulating both paracrine and endocrine IGF-I (20, 46, 47). We, in turn, have previously shown that mammary involution, involving massive apoptotic cell death at the end of lactation (48) is the result of an increase in the production of IGFBP-5 (15, 16, 17). We proposed that this binding protein serves to inhibit IGF-mediated cell survival, which has been described in several model systems (10, 11, 13, 14). We have confirmed this proposal by demonstrating, for the first time, that administration of exogenous IGFBP-5 during the periparturient period leads to impaired mammary gland development and function. Mammary gland weight, DNA content, and proportion of parenchymal tissue were all significantly decreased by exogenous IGFBP-5. In addition, these animals produced significantly less milk than untreated animals during the first 5 d of lactation.

As PRL is the major factor responsible for inhibiting the synthesis of IGFBP-5 by the mammary epithelium (17), we anticipated increased expression of IGFBP-5 in PRLR+/- mice and enhanced cell death as a result. In our model, mammary development is dependent upon the relative balance between IGF-I (survival) and IGFBP-5 (apoptosis). We therefore hypothesized that mammary development in the PRLR+/- mouse might be rescued by increasing circulating concentrations of IGF-I, and we used GH treatment to achieve this. Our findings clearly add further weight to this hypothesis, as IGFBP-5 concentrations were indeed increased 3-fold in PRLR+/- mice, whereas GH treatment prevented this increase in IGFBP-5 concentrations and, at the same time, increased by 3-fold the concentration of IGF-I. This alteration in the ratio between IGF-I and IGFBP-5 was accompanied by a restoration of mammary development in PRLR+/- mice given GH. Consistent with the impairment in mammary development, we demonstrated that plasmin concentrations were elevated in PRLR+/- mice, indicative of a breakdown of the extracellular matrix (32, 33), and GH treatment also prevented this. We have previously shown similar increases in plasmin activity during involution of the mammary gland at the end of lactation and that GH could partially prevent this (34). It must be recognized, however, that other possible mechanisms may contribute to this effect of GH in the PRLR+/- mouse. For example, GH has been shown to increase estrogen receptor expression in the rat mammary gland, an effect not reproduced by des(1–3)IGF-I (49), and it exhibits a pretranslational control on epidermal growth factor receptors (50). In addition, and perhaps of greater relevance to this particular model, GH is well known to increase PRL receptor expression in the liver (51). These possibilities are obviously worthy of further study.

The present study confirms and significantly extends our original findings regarding the phenotype of the PRLR+/- mouse (5), revealing some of the mechanistic aspects involved in the developmental failure evident in these animals. The improved mammary development could have been due to reduced apoptosis, increased rates of proliferation, or a combination of both. Our results do not allow us to distinguish between these possibilities, but the fact that the changes in plasmin and IGFBP-5 are so reminiscent of mammary involution, which involves increased apoptosis (15, 16, 17, 32, 33, 34), suggests that alterations in the rate of apoptosis are likely to be at least part of the explanation of these effects.

Much of the mammary gland on d 2 of lactation was arrested in PRLR+/- mice, at a stage similar to that on d 14 of pregnancy. Somewhat to our surprise, however, when we examined the metabolic activity of the glands in these animals, it was clear that the small number of alveolar structures that did develop showed a reasonable degree of induction of mRNAs for the major milk protein ß-casein and for ACC-{alpha}, including the characteristic increase in the ratio of short to long forms of the ACC-{alpha} mRNAs (52). This clearly implied that some degree of lactogenesis was being induced. However, in all cases the milk protein content or enzyme activity of the mammary gland in PRLR+/- mice was induced to a much lesser extent than was the mRNA, suggesting decreased translational efficiency or protein stability. In our attempt to rescue the phenotype of the PRLR+/- mouse, we treated animals with GH during the last 7 d of pregnancy. GH dramatically improved mammary development, resulting in a mammary fat pad entirely filled with alveolar structures. In our original studies we proposed that the phenotype of the PRLR+/- mouse involved three defects: 1) deficient development postpubertally, 2) deficient development during pregnancy, and 3) deficient postpartum development of milk synthetic capacity. The last of these could simply have been a consequence of the impaired alveolar development before parturition. However, the present study clearly demonstrates that some form of PRLR deficiency does indeed extend into lactation, because even though GH normalized mammary development to the point where, morphologically, the glands were indistinguishable from those of wild-type animals, the capacity to synthesize and secrete milk was considerably impaired in GH-treated PRLR+/- mice. Indeed, when we examined various metabolic parameters in the mammary glands of GH-treated animals, we discovered that, rather than reducing the phenotypic deficiencies of the PRLR+/- mouse, they actually exacerbated them. Most striking of all was the expressed activity of acetyl-CoA carboxylase, which was less than 5% of wild-type values when expressed per cell. Although the mammary glands of GH-treated animals contained many more cells than PRLR+/- mice not treated with GH, the total capacity to synthesize fatty acids was still well below 10% of that in wild-type animals. As more than 60% of milk energy is derived from fat in rodents (26), an impaired ability to synthesize milk lipid, even if milk volume was maintained to a reasonable degree, would ensure that pups would not thrive compared with wild-type pups. This was indeed the case.

Our findings are consistent with data showing that GH is important in terms of mammary development during pregnancy (19). Furthermore, our data support the findings that GH is only weakly lactogenic (23) and can only partially substitute for PRL in terms of galactopoiesis (established milk synthesis), typically maintaining milk secretion and steady state levels of casein mRNAs at 40% of the levels induced by PRL (16, 25, 26). Acetyl-CoA carboxylase activity in the mammary gland is even more dependent upon PRL, as GH can only maintain levels at about 20% of control values in PRL-deficient rats (27). We therefore propose that GH treatment, possibly acting indirectly via increased concentrations of IGF-I, induces a proliferative state, rather than the differentiative state that normally occurs at parturition.

The deficiency in mammary development could be attributed to the fact that only one allele of the PRLR is functional, as demonstrated by reduced PRL binding in the liver of +/- mice. PRL binding was thus determined as previously described (4) and averaged 13.5% in wild-type liver microsomes compared with 5.7% in PRLR+/- liver. We previously suggested that there may be a threshold level of receptor expression required to induce mammary development. Cytokines such as PRL and GH have signal transduction pathways that involve dimerization of two receptors (53, 54). It is conceivable that decreases in PRLR density of 50% may be sufficient to reduce the possibilities of PRLR dimerization occurring before 1:1 complexes dissociate. Alternatively, this impairment of signaling may reflect the fact that the PRLR associates with Janus kinase 2 independent of its occupancy by PRL (55), which contrasts with that of GH receptor, where Janus kinase 2 association does not occur until GH binding occurs (see Ref. 56 for review). We have previously shown that PRL signal transduction can be inhibited by suppressor of cytokine signaling expression (57). Recent findings of Lindeman et al. (58) shed light on the defect in PRLR+/- mice by demonstrating that deletion of one allele of the suppressor of cytokine signaling gene results in normal mammary development, suggesting that increasing the lactogenic signal, by reducing the inhibition of PRL signaling, is also effective.

In summary, our data illustrate that the abnormal mammary gland development that occurs in young PRLR+/- mice includes increased expression of the proapoptotic molecule IGFBP-5 and plasmin production. Despite the abnormal mammary development in PRLR+/- mice, a surprising degree of differentiation still occurred during the peripartum period. We have also demonstrated that alveolar development can be rescued by GH treatment during late pregnancy, probably acting via increased IGF-I synthesis and suppression of IGFBP-5 and plasmin generation. However, despite full lobuloalveolar development, the synthetic capacity of the mammary gland remained impaired, indicating that GH cannot fully replace PRL in this respect. Indeed, GH actually exacerbated the metabolic defects in PRLR+/- mice, possibly by maintaining a proliferative, rather than a differentiative, environment during the peripartum period. Furthermore, we were able to demonstrate that IGFBP-5 could impair mammary development, suggesting that it is an important factor in the impaired development of the PRLR+/- mouse mammary gland, and this supports the proposal that IGF-I is an important developmental factor for the mammary gland.

Our data add further weight to the concept that GH plays an important role in mammary growth, but plays a lesser role than PRL in driving metabolic processes in lactating mammary gland. The nature of the PRL deficiency in PRLR+/- mice remains intriguing, as it results from the expression of half the normal number of PRLR in the mammary gland. This implies that intracellular signaling is compromised by the reduced number of PRLR molecules available. The mechanisms involved in this impairment are also clearly worthy of further study.


   Acknowledgments
 
We thank M. Gardner and S. Boumard for skilled technical assistance, D. Blatchford for antibodies to mouse caseins and WAP, and Dr. S. Brocklehurst for the statistical analyses.


   Footnotes
 
This work was supported in part by grants from Institut National de la Santé et de la Recherche Médicale, Organon FARO 61/subv.99, Ministère de l’Education Nationale de la Recherche et de la Technologie (1A010G), ARC subv. 9952, and the Scottish Executive Environment and Rural Affairs Department and by a Royal Society Fellowship (to D.J.F.).

1 N.B. and D.J.F. are co-senior authors. Back

Abbreviations: ACC-{alpha}, Acetyl-coenzyme A carboxylase-{alpha};acetyl-CoA, acetyl-coenzyme A; IGFBP, IGF-binding protein; PRL, prolactin; PRLR, PRL receptor; PRLR ko, PRL receptor knockout; WAP, whey acid protein.

Received October 19, 2001.

Accepted for publication July 22, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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