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BOR - Papers in Press, published online ahead of print August 3, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.043992
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BIOLOGY OF REPRODUCTION 73, 1116–1125 (2005)
DOI: 10.1095/biolreprod.105.043992
© 2005 by the Society for the Study of Reproduction, Inc.

Research Article

Overexpression of Des(1–3) Insulin-Like Growth Factor 1 in the Mammary Glands of Transgenic Mice Delays the Loss of Milk Production with Prolonged Lactation1

Darryl L. Hadsell 2 35 , Daniel T. Torres 3, Nicole A. Lawrence 3, Jessy George 3, Albert F. Parlow 6, Adrian V. Lee 45 , and Marta L. Fiorotto 3

USDA/ARS Children's Nutrition Research Center,3 Department of Pediatrics The Breast Center, Department of Medicine,4 and Department of Molecular and Cellular Biology,5 Baylor College of Medicine, Houston, Texas 77030 National Hormone Peptide Program,6 Harbor-UCLA Medical Center, Torrance, California 90509

ABSTRACT

During prolonged lactation, the mammary gland gradually loses the capacity to produce milk. In agricultural species, this decline can be slowed by administration of exogenous growth hormone (GH), which is believed to act through insulin-like growth factor 1 (IGF1). Our previous work demonstrated delayed natural mammary gland involution in des(1–3)IGF1-overexpressing transgenic mice (Tg[Wap-des{1–3}IGF1]8266 Jmr), hereafter referred to as WAP-DES mice. The present study tested the hypothesis that overexpressed des(1–3)IGF1 would delay the loss of milk production during prolonged lactation. Accordingly, we examined lactational performance in WAP-DES mice by artificially prolonging lactation with continual litter cross-fostering. Over time, lactational capacity and mammary development declined in both WAP-DES and control mice. However, the rate of decline was 40% slower in WAP-DES mice. Mammary cell apoptosis increased by 3-fold in both groups during prolonged lactation but was not different between genotypes. Plasma concentrations of murine IGF1 were decreased in WAP-DES mice, while those of the transgenic human IGF1 were elevated during prolonged lactation. Phosphorylation of the mammary IGF1 receptor was increased in the WAP-DES mice, but only during prolonged lactation. Plasma prolactin decreased with prolonged lactation in nontransgenic mice but remained high in WAP-DES mice. The WAP-DES mice maintained a higher body mass and a greater lean body mass during prolonged lactation. These data support the conclusion that overexpressed des(1–3)IGF1 enhanced milk synthesis and mammary development during prolonged lactation through localized and direct activation of the mammary gland IGF1 receptor and through systemic effects on prolactin secretion and possibly nutrient balance.

insulin-like growth factor receptor, kinases, lactation, mechanisms of hormone action, prolactin, persistence, transgenic

INTRODUCTION

Maintenance of milk synthesis during lactation depends on endocrine and paracrine signals generated in response to suckling and milk removal [1–4]. In the absence of milk removal or with decreased milk removal as occurs during weaning, the gland undergoes a dramatic remodeling process termed involution. Mammary gland involution is characterized by cessation of milk synthesis followed by a wave of cellular apoptosis and tissue remodeling [2]. Continuous removal of milk from the gland can prolong lactation and prevent involution. Despite this, however, the rate of milk synthesis during prolonged lactation declines over time, suggesting that degenerative processes are at work within the secretory epithelium. In agriculturally important species as well as in rodents, this decline in milk synthesis may be due to both decreased mammary cell number and decreased synthetic capacity of the secretory cells [5–8]. The extent to which this degenerative process is delayed, a property referred to as persistence, is of great importance to the dairy industry. In addition, an understanding of the underlying physiological processes that regulate lactation persistence may also have applications to human lactation, fertility, and perhaps even aging.

The regulation of lactation persistence has been discussed in several recent reviews [9–11]. The factors involved in this process include frequency of milk removal, photoperiod, and reproductive status. In agricultural species, decreasing the daily frequency of milk removal accelerates the rate at which milk synthesis decreases, whereas increasing this frequency has the opposite effect [12, 13]. In both circumstances, changes occur in the size of the epithelial cell population within the gland [4, 14]. Observations have been collected in breast-feeding women, which also suggest that a similar phenomenon may operate in humans [15]. The regulation of milk synthesis during prolonged lactation by milk removal involves both local signals within the gland as well as endocrine signals originating in the pituitary. In rats, both prolactin and glucocorticoids have been demonstrated to enhance milk yield during prolonged lactation when given exogenously, and previous studies have demonstrated that blood prolactin and corticosterone concentrations decrease during progression through lactation, while those of LH and FSH actually increase [7, 16–20]. Intriguingly, studies in rodents have also demonstrated enhanced milk synthesis during prolonged lactation in response to ovariectomy or treatment with progesterone, leading to the suggestion of a functional link between resumption of estrus cycles and loss of milk synthesis [20, 21]. In addition, both oxytocin and GH, if given exogenously, improve lactation persistence in dairy cows [22, 23].

Although numerous studies support the hypothesis that IGF1 may mediate some of the effects of GH on lactation [24], the capacity of IGF1 to enhance lactation persistence has not been directly tested. In previously published studies with the Tg(Wap-des[1–3]IGF1)8266 Jmr mouse model (WAP-DES), a strain engineered to overexpress human des(1–3)IGF1 in the mammary gland under the control of a milk protein gene promoter, we demonstrated that overexpression of this analog of IGF1, which has reduced binding to IGF binding proteins, inhibited mammary involution during late (Days 18–20 postpartum) lactation [25, 26]. This effect, though associated with increased mammary gland wet weight and diminution of mammary cell apoptosis, was found to occur in the presence of only modest increases in the phosphorylation of key IGF1-dependent signaling proteins.

The direct effects of IGF1 in the mammary gland are believed be mediated through the type 1 IGF receptor (IGF1R)-dependent phosphorylation of insulin receptor substrate (IRS) proteins followed by subsequent phosphorylation and activation of the serine/threonine kinases, MAPK3/1 (ERK1/2), and AKT. The support for this idea comes primarily from cell culture studies and has been discussed in several recent reviews [27, 28]. In addition, recent work conducted in our laboratory has demonstrated rapid and pronounced induction of the phosphorylation of IRS1, MAPK1 and 3, and AKT within the mammary glands of virgin female mice in response to intravenous injections of IGF1, suggesting that these proteins are mediators of IGF1 action in the mammary gland within the context of a normal animal [29]. The studies described in this paper tested the hypothesis that overexpression of des(1–3) IGF1 during prolonged lactation would activate IGF1-dependent cell survival pathways, inhibit the loss of mammary cells, and thereby enhance milk synthesis during prolonged lactation. Accordingly, lactation performance and mammary gland development along with indicators of IGF and prolactin signaling were compared during prolonged lactation in nontransgenic and WAP-DES mice.

MATERIALS AND METHODS

Experimental Animals

All animals were studied in accordance with procedures outlined in the NIH Guide to Care and Use of Experimental Animals. These experiments were approved by the Baylor College of Medicine Animal Care and Use Committee. The transgenic mice used in these studies were the previously described Tg(Wap-des[1–3]IGF1)8266 Jmr strain of mice that overexpress human des(1–3)IGF1 in the mammary gland under the control of the promoter for the whey acidic protein gene [25, 26]. These mice will be hereafter referred to as WAP-DES mice. The animals were analyzed in their first lactation, and none were concurrently pregnant during the course of these studies. A total of three studies were done for comparison of lactation performance in nontransgenic (n = 8) and WAP-DES (n = 14) mice during both early and prolonged lactation. For all three studies, each dam received a cross-fostered litter of 10 1-day-old CD-1 pups beginning on Day 1 postpartum to control for pup genotype and litter size. In addition, litter weights were equalized across all dams. In the first study, lactation was prolonged out to 42 days by cross-fostering 7-day-old CD-1 pups onto each dam every 7 days, beginning on Day 14 postpartum. Dam weight and litter weight gains were recorded for each 7-day period, and milk samples were collected out to Day 42 pospartum. In the second and third studies, litter weights were followed to Day 35 postpartum, and tissues were harvested on Day 37. Body composition of the dams was measured in the second and third studies on Day 36 by scanning each animal once with a Piximus (Lunar Corp.) dual x-ray absorptiometer (DXA), as previously described [30]. A fourth cohort of nontransgenic (n = 13) and WAP-DES (n = 4) dams was used to provide samples on Day 8 postpartum. Mammary glands were harvested on Days 8, 37, and 42 postpartum to provide samples from both early and prolonged lactation. At the time of harvesting, wet weights were recorded on each of the number four mammary glands. The glands were then either flash frozen in liquid nitrogen and stored at –80°C for further analysis or frozen in OCT embedding medium for cryosectioning. A subset of dams from this study were also given an intraperitoneal injection (100 mg/kg BW) of bromodeoxyuridine (BrdU) (Sigma Chemical Corp.) 2 h before euthanasia to label mammary cells in S-phase of the cell cycle. In studies 2 and 3, plasma samples were prepared from trunk blood collected at the time of euthanasia, and in study 3, pituitaries were collected.

Milk and Tissue Analysis

Milk samples were analyzed for lactose, nitrogen, fat, and water as previously described [31]. Epithelial content of the mammary tissue was determined by segmentation analysis of images captured from hematoxylin-eosin-stained mammary tissue as previously described [31]. Mammary cell proliferation was measured in frozen tissue sections by staining for BrdU incorporation with a FITC-conjugated anti-BrdU antibody (Becton Dickinson). Mammary cell death was detected by fluorescent TUNEL staining as previously described [32]. Enumeration of positive cells was accomplished by counting 50 randomly selected 40x fields. This was equivalent to counting about 10000–15000 cells per sample. All specimens were counterstained with the nuclear dye TOPRO 3 (Molecular Probes). Specimens were then visualized using an Olympus FV300 confocal microscope (Olympus America).

Hormone Measurements

The plasma concentrations of both human and murine IGF1 were measured in samples collected at Days 8 and 37 postpartum. The IGF1 assays were both ELISA-based assays. The human IGF1 assay was a nonextraction IGF1 ELISA (Diagnostic Systems Laboratories Inc.) with a sensitivity of 20 ng/ml and an intra-assay coefficient of variation of 5%–9%. Murine IGF1 was measured using a rat/mouse-specific immunoenzymometric assay (Immunodiagnostic Systems Inc.) with a sensitivity of 82 ng/ml and an intra-assay coefficient of variation of 5%–7%. For analysis of plasma prolactin, samples were shipped on dry ice to the National Hormone and Pituitary Program (Torrance, CA). The RIA immunoreagents are distributed to researchers on request by the National Institute of Diabetes and Digestive and Kidney Diseases, National Hormone and Pituitary Program. Analysis of pituitary prolactin content was accomplished likewise on whole mouse pituitaries homogenized in 1 ml of 0.01 M sodium bicarbonate buffer (pH 8.0).

Western Blotting

Total tissue protein extracts of mammary tissue were prepared from 50 mg of tissue, and Western blotting was conducted as previously described [31]. Briefly, blots were prepared using PROTRAN nitrocellulose (Schleicher & Scheull). Detection was based on enhanced chemiluminensence using Supersignal westpico chemiluminescent substrate (Pierce), an HRP-conjugated donkey anti-rabbit at a dilution of 1:2000 (Amersham Biosciences), and BioMax MR film (Kodak). Phosphorylation of the insulin receptor (INSR) and IGF1R was measured using an antibody (1:1000 dilution) specific for triple-phosphorylation of the catalytic-domain tyrosines: 1158, 1162, and 1163 for INSR and 1131, 1135, and 1136 for IGF1R, respectively (Biosource International). Phospho-AKT was measured using an antibody (1:1000 dilution) to phospho-Ser473, which reacts with all three AKT isoforms (Cell Signaling Technology). Phospho-MAPK3/1 (ERK1/2) was measured using an antibody (1:1000 dilution) that detects dual phosphorylation (dp) of Thr202 and Tyr204 (Cell Signaling Technology). Phosphorylation of STAT5 was detected using an antibody (1:1000 dilution) to phospho-Tyr694 (Santa Cruz Biotechnology). Phosphorylation of IRS1 was measured by Western blotting with an antibody specific for phospho-Tyr612 (Biosource International). Total amounts of each of IRS1, IRS2, AKT1, and MAPK3/1 were also measured by Western blotting as previously described [26]. As positive controls for induction of phosphorylation, liver, kidney, or mammary tissue extracts were prepared from either virgin or lactating female mice injected with 0.1 ml of saline containing 5 or 50 µg of either recombinant human LR3-IGF1 (Gropep Ltd) or bovine insulin (Sigma) [29]. Equality of protein loading among the samples was tested two ways. First, the samples were run on parallel gels that were subsequently stained with Coumassie blue. Second, parallel blots were prepared and probed with an antibody (1:3000) to the housekeeping protein ß-tubulin (Abcam Inc.). Densitometry data were collected using a Molecular Dynamics personal densitomer SI.

Data Analysis

Litter weight gain, dam weight, and milk composition data were analyzed using the mixed-models procedure of SPSS (version 12.01 for Windows) with genotype as the fixed variable and day postpartum as a repeated measure within each dam. Data for mammary epithelial area, plasma IGF1, and plasma prolactin was analyzed as a 22 factorial design using the GLM procedure of SPSS. Body composition data were analyzed as a one-way ANOVA assuming unequal variance between genotypes. Pituitary weight and prolactin content were analyzed by simple unpaired t-test assuming unequal variances. Specific hypotheses concerning genotype effects within a specific time or main effects of genotype or time were tested using orthogonal contrast. Western blotting data were analyzed as a 22 factorial design using day and genotype as independent variables. Densitometry data from the Coumassie blue-stained gels were used to adjust the Western blot data for variations in loading. All data are presented as LS means ± SEM. Differences were considered statistically significant at {alpha} = 0.05.

RESULTS

Previous studies on transgenic models of IGF1 overexpression in the mammary gland have demonstrated modest, if any, enhancement of milk yields during early lactation [33–35]. However, because we observed delayed mammary involution in the WAP-DES transgenic mice during the latter part of a normal lactation [26], we chose to determine the effects of the transgene on milk production during artificially prolonged lactation. To determine the effect of overexpressed des(1–3)IGF1 on lactation performance during prolonged lactation, groups of nontransgenic and WAP-DES mice were initially cross-fostered on Day 1 postpartum with 10 1-day-old pups and then placed on a weekly cross-fostering protocol beginning on Day 14 postpartum. Lactation capacity was assessed from the weekly gain in litter weight (Fig. 1 A). This analysis demonstrated that lactation capacity was highest on Day 14, declined gradually by Day 21, and then decreased much more rapidly by Day 28 postpartum. In nontransgenic dams, lactation capacity by Day 35 postpartum was insufficient to support weight gain of their litters. In WAP-DES dams, lactation capacity also declined by Day 28 postpartum. However, WAP-DES dams were still capable of supporting significantly greater (P < 0.05) litter weight gain than nontransgenic dams on both Day 28 and Day 35 postpartum. This increased lactational capacity was associated with increased (P < 0.05) wet weight of the number 4 mammary glands in WAP-DES dams compared to nontransgenic dams on Day 37 postpartum (336 ± 12 versus 295 ± 9 mg, respectively). By Day 42 postpartum, capacity to support litter weight gain was equally limited in both groups. These results support the conclusion that overexpression of des(1–3)IGF1 in the mammary gland delayed but did not abolish the loss of milk production that occurred during prolonged lactation.


Figure 1
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  		FIG. 1. Lactation performance and mammary gland development is enhanced in WAP-DES mice during prolonged lactation. The ability of nontransgenic (black circles) and WAP-DES (gray circles) dams to support the weight gain of cross-fostered litters was followed out to 42 days postpartum (A). Lactation was artificially prolonged by cross-fostering litters weekly beginning on Day 14 postpartum. Each symbol represents the LS mean ± SEM litter weight gain during the preceding week. There were eight nontransgenic and 14 WAP-DES dams, except on Day 42, when there were only four and five, respectively. Percent epithelium (B) was measured in images captured from hematoxylin-eosin-stained sections of mammary tissue collected from nontransgenic (black boxes) and WAP-DES (gray boxes) dams on Days 8, 37, and 42 postpartum. Each bar represents the LS mean ± SEM for five and four, four and eight, and four and five nontransgenic and WAP-DES dams on Days 8, 37, and 42, respectively. Asterisks indicate a significant difference between nontransgenic and WAP-DES dams (P < 0.05)

To determine if differences in the loss of milk production during prolonged lactation could be accounted for by changes in glandular development, the area percentage occupied by secretory epithelium was measured in images of hematoxylin-eosin-stained mammary tissue sections prepared from samples collected at Days 8, 37, and 42 postpartum (Fig. 1B). This analysis demonstrated that epithelial content decreased (P < 0.0001) during prolonged lactation in both groups. Mammary epithelial content of WAP-DES dams decreased at a slower rate than that of nontransgenic dams and was significantly greater (P < 0.005) on both Day 37 and Day 42 postpartum. This result supports the suggestion that the mammary epithelial cell loss during prolonged lactation was slower in the WAP-DES dams than in the nontrangenic dams.

To determine if the developmental changes observed in the WAP-DES dams could be accounted for by changes in cell proliferation or cell death, mammary tissue sections were stained for BrdU incorporation or a marker of apoptosis (Fig. 2). The percent BrdU incorporation was similar among nontransgenic and WAP-DES mice on Day 8 postpartum (Fig. 2A). On Day 37 postpartum, BrdU incorporation appeared to be higher in mammary tissue from WAP-DES dams than that observed in nontransgenic dams. This difference, however, only approached statistical significance (P < 0.09). When averaged over both genotypes, the percent mammary cell apoptosis (Fig. 2B) was about 3-fold higher (P < 0.05) at Day 37 than at Day 8 postpartum (1.54 ± 0.40% versus 0.46 ± 0.12%, respectively). Percent apoptosis was similar, however (P > 0.05), among nontransgenic and WAP-DES dams. The results support the conclusion that the rate of mammary cell death increases with prolonged lactation. The results also suggest that overexpression of IGF1 within the mammary gland during prolonged lactation does not dramatically alter apoptosis but may enhance cell proliferation.


Figure 2
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  		FIG. 2. Mammary cell proliferation and apoptosis during early and prolonged lactation. Bromodeoxuridine positive nuclei (A) were detected using immunofluoresence and counted for the percentage of mammary cells in S-phase among nontransgenic (black boxes) and WAP-DES (gray boxes) mice on Day 8 or Day 37 postpartum. Apoptotic cells were detected using fluorescent TUNEL staining and counted to measure cell death (B). For both assays the percentage of positive cells was calculated based on total cell counts in specimens counterstained with TOPRO 3. Each bar represents the LS mean ± SEM for 12 and 4 and 10 and 11 nontransgenic and WAP-DES mice at Days 8 and 37, respectively. The number sign indicates a trend (P < 0.09) toward a statistically significant increase in BrdU labeling. Apoptosis was higher on Day 37 than on Day 8 postpartum (P < 0.05)

The growth of the suckling neonate depends both on total milk intake and on the nutrient composition of milk. To determine the degree to which our lactation performance measurements were related to changes in milk nutrient composition, milk samples were collected from nontransgenic and WAP-DES dams and analyzed for lactose, water, nitrogen, and fat (Fig. 3). Milk composition was similar in both genotypes throughout the course of the study. When averaged over both genotypes, a main effect (P < 0.05) of day postpartum was observed for both water and lactose concentrations. However, though these changes were statistically significant, they were modest. Neither fat nor nitrogen content changed appreciably with prolonged lactation.


Figure 3
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  		FIG. 3. Milk lactose and water varied over the course of lactation, but milk composition was similar among nontransgenic and WAP-DES dams. Milk water (A), lactose (B), nitrogen (C), and fat (D) were measured among nontransgenic and WAP-DES mice throughout the course of a prolonged lactation. Concentrations of all of these components were similar among the two genotypes. Each bar represents the LS mean ± SEM for two to five mice. A main effect of days postpartum was detected for both water (P < 0.05) and lactose (P < 0.005)

The expression of human IGF1 in the WAP-DES transgenic model has been previously shown to be limited to the lactating mammary gland. In addition, our early work with these mice failed to detect any of the transgene protein in the circulation at Day 10 postpartum [25]. Therefore, we initially reasoned that the WAP-DES dams would have a similar body weight as nontransgenic dams. To our surprise, the WAP-DES dams weighed 11% more (P < 0.05) than nontrangenic dams by the end of the study (Fig. 4A). This difference was corroborated by body composition measurements (Fig. 4B) that demonstrated that the WAP-DES dams had 13% greater (P < 0.05) lean body mass than nontransgenic dams. These data suggested to us that the WAP-DES transgene was capable of eliciting systemic effects during prolonged lactation.


Figure 4
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  		FIG. 4. Total body weight and lean body mass were greater in WAP-DES dams at 37 days of lactation. Body weight (A) was recorded weekly for nontransgenic (black circles) and WAP-DES (gray circles) dams. Each symbol represents the LS mean ± SEM for 9 and 13 nontransgenic and WAP-DES mice, respectively. Body composition (B) in nontransgenic (black boxes) and WAP-DES (gray boxes) dams was measured on Day 36 postpartum using a PIXImus mouse densitometer. Each bar represents the LS mean ± SEM for 9 and 14 nontransgenic and WAP-DES mice, respectively. Asterisks indicate statistical significance (P < 0.05)

To determine if the body weight and composition effects that we observed in the WAP-DES dams were associated with changes in circulating IGF1 levels, we measured both human IGF1 and murine IGF1 in plasma samples collected from the dams at Days 8 and 37 postpartum (Fig. 5A). The plasma concentration of human IGF1 was below the limit of detection in our assay for both the WAP-DES and the nontransgenic dams on Day 8 postpartum. However, plasma human IGF1 concentrations in WAP-DES dams reached 432 ± 72 ng/ml at Day 37 postpartum. The plasma concentration of murine IGF1 was decreased (P < 0.05) with prolonged lactation (332 ± 8 and 295 ± 8 ng/ml for Days 8 and 37, respectively) and was consistently lower overall (P < 0.05) in the WAP-DES dams than nontransgenic dams (285 ± 9 and 333 ± 7 ng/ml, respectively). These data indicate that during prolonged lactation the des(1–3)IGF1 expressed by the WAP-DES promoter was able to leak into the circulation from the mammary gland and raise total circulating plasma IGF1 concentrations.


Figure 5
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  		FIG. 5. Plasma IGF1 concentrations are altered in WAP-DES mice during prolonged lactation. Concentrations of transgenically produced (A) human IGF1 and endogenous (B) murine IGF1 were measured in plasma samples collected from nontransgenic (black boxes) and WAP-DES (gray boxes) dams on Days 8 and 37 postpartum of an artificially prolonged lactation. Each bar represents the LS mean ± SEM for 12 and 4 and 9 and 12 nontransgenic and WAP-DES dams on Days 8 and 37, respectively. Significant main effects on murine IGF1 levels were observed for both genotype (P < 0.0001) and day (P < 0.05). On Day 8 postpartum, human IGF1 was below the level of detection (20 ng/ml) in the assay. Asterisk indicates a statistically significant (P < 0.05) decrease in mIGF1 on Day 8 postpartum

To determine if differences in prolactin contributed to the enhanced performance of the WAP-DES mice, we measured prolactin concentrations in plasma (Fig. 6A) and pituitary (Fig. 6B). Overall, the plasma concentration of prolactin decreased (P < 0.01) with prolonged lactation (229 ± 35 and 52 ± 30 ng/ml for Days 8 and 37, respectively). There was also a genotype-by-day interaction (P < 0.04) in that plasma prolactin remained 2.7-fold higher (P < 0.01) at Day 37 in WAP-DES dams than that observed in nontransgenic dams (Fig. 6A). Although there was no difference in pituitary weight, prolactin content of the pituitary was higher (P < 0.05) in the WAP-DES dams than nontransgenic dams (Fig. 6B). These results suggest that enhanced performance in WAP-DES dams during prolonged lactation may have been partially mediated through increased prolactin secretion.


Figure 6
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  		FIG. 6. Plasma prolactin decreases with prolonged lactation but remained higher in WAP-DES dams than nontransgenic dams. Plasma prolactin concentration (A) was measured in plasma samples prepared from trunk blood of nontransgenic (black boxes) and WAP-DES (gray boxes) dams on Days 8 and 37 postpartum. Each bar represents the mean ± SEM for 13 and 4 and 10 and 15 nontransgenic and WAP-DES dams on Days 8 and 37, respectively. A significant main effect of day postpartum and a significant day-by-genotype interaction was observed (P < 0.05). Pituitary weight (black boxes) and prolactin (gray boxes) content were measured in nontransgenic and WAP-DES dams at Day 37 postpartum (B). For pituitary weight, each bar represents LS mean ± SEM for seven and five nontransgenic and WAP-DES dams, respectively. For prolactin content, each bar represents the data from four and three nontransgenic and WAP-DES dams, respectively

To determine if intracellular signaling pathways for IGF1 were altered by prolonged lactation or overexpression of IGF1, the phosphorylation of key signaling proteins within the mammary glands of nontransgenic and WAP-DES dams was analyzed by Western blotting. As positive controls, we injected fasting female mice with either insulin or LR3-IGF1. Injection of lactating mice with 5 µg of insulin (Fig. 7A, lane 1) or IGF1 (Fig. 7A, lane 2) induced phosphorylation of INSR or IGF1R in the mammary gland, as shown by the presence of distinct phosphoprotein bands on blots probed with the pINSR/pIGF1R antibody. Injection with insulin produced a band that migrated slightly below the 97-kDa marker used with these studies. Injection with LR3-IGF1 produced two to three bands, depending on the dose administered. At a dose of 5 µg, bands were produced that migrated at and slightly above the 97-kDa marker (data not shown). At the dose of 50 µg, LR3-IGF1 produced three bands in the mammary gland, one that migrated slightly above, one that migrated with, and one that migrated slightly below the 97-kDa marker (Fig. 7A, lane 2). The band that migrates below 97 kDa is probably INSRß because this band is also induced by insulin. The band at 97 kDa is of the approximate size for IGF1Rß, while the larger band is consistent with the report of a larger, fetal form of IGF1Rß that has been described previously [36]. Both insulin and LR3-IGF1 were also found to induce the phosphorylation of AKT and MAPK3/1 within the mammary gland (data not shown).


Figure 7
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  		FIG. 7. A) Phosphorylation of IGF1Rß is increased in WAP-DES mice at Day 37 postpartum. Mammary tissue extracts were prepared from nontransgenic (–) and WAP-DES (+) mice on Days 8 and 37 of lactation. The total number of samples analyzed was 12 and 4 and 8 and 13 nontransgenic and WAP-DES dams on Days 8 and 37, respectively. For each lane, 20 µg of extract protein were (100 µg for AKT and pINSR/pIGF1R) loaded. The resulting gels were then blotted and the membranes probed for pINSR/pIGF1R, phospho-AKT, phospho-MAPK3/1, total AKT1, total MAPK3/1, IRS1, and IRS2. Control mammary gland extracts generated for the pINSR/pIGF1R blot were prepared by injecting female mice with either saline (lane 3), saline + 5 µg insulin (lane 1), or saline + 50 µg LR3-IGF1 (lane 2). Densitometry data are shown for pINSR/pIGF1R (B) phospho-IRS1 and total IRS1 (C) and IRS2 (D). Refer to the graph for the legend. Each bar represents the LS mean ± SEM for 12 and 4 and 9 and 14 nontransgenic (NT) and WAP-DES (WD) dams at Days 8 and 37, respectively. Neither the phosphorylation nor the expression of AKT or MAPK3/1 was influenced by day or genotype (densitometry data not shown)

Comparison of signaling protein phosphorylation in nontransgenic and WAP-DES mice on Days 8 and 37 postpartum demonstrated that overexpression of des(1–3)IGF1 in the mammary gland increased phosphorylation of IGF1Rß at Day 37 postpartum (Fig. 7A, lane 7). Densitometric analysis of the pINSR/pIGF1R blots (Fig. 7B) demonstrated that IGF1Rß phosphorylation was increased 3-fold in mammary tissue from the WAP-DES mice but only on Day 37 postpartum. However, neither the phosphorylation of INSRß nor that of the putative fetal form were influenced by genotype or stage of lactation. Phospho-IRS1 and total IRS1 and IRS2 were decreased in the WAP-DES dams (P < 0.05) but only on Day 8 postpartum (Fig. 7, C and D). Neither phosphorylation nor expression of AKT or MAPK3/1 was affected by genotype or stage of lactation. These results support the conclusion that chronic overexpression of des(1–3)IGF1 in the mammary gland during prolonged lactation was effective at activating the mammary IGF1R but had little effect on the phosphorylation of the downstream signaling proteins IRS1, MAPK3/1, or AKT.

Because plasma prolactin decreased with prolonged lactation and because the WAP-DES mice had higher concentrations of plasma prolactin at Day 37 than their nontransgenic siblings, we measured the levels of both phospho-STAT5 and total STAT5 by Western blotting (Fig. 8). Both phospho-Tyr694-STAT5 and total STAT5 were decreased in mammary glands from mice on Day 37 relative to that observed for mice on Day 8 postpartum (Fig. 8A). Densitometry (Fig. 8B) demonstrated that the levels of both phospho-Tyr694 and total STAT5 were lower on Day 37 than Day 8 postpartum (P < 0.005). The ratio of phospho-STAT5 to total-STAT5 was also decreased (P < 0.01) between Day 8 and Day 37 postpartum (0.672 ± 0.064 and 0.443 ± 0.047, respectively). There was also a modest main effect (P < 0.05) of genotype on this ratio, which was 35% higher in mammary tissue from WAP-DES dams compared to that of nontransgenic dams (0.640 ± 0.063 versus 0.475 ± 0.049, respectively). Mammary abundance of the housekeeping protein ß-tubulin was similar among both genotypes and was not influenced by day postpartum (Fig. 8A). These data suggest that the decrease in plasma prolactin that was observed on Day 37 postpartum was associated with decreased activation of the JAK-STAT pathway within the mammary gland.


Figure 8
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  		FIG. 8. STAT5 phosphorylation and total levels of STAT5 are decreased with prolonged lactation. Mammary tissue extracts were prepared from nontransgenic (–) and WAP-DES (+) mice on Day 8 of a normal lactation and on Day 37 of prolonged lactation. Each lane contained 20 µg of mammary tissue extract from an individual mouse. Following electrophoresis and blotting, the resulting blots were probed with an antibody of the phosphorylated Tyr694 of STAT5 and then reprobed with an antibody to total STAT5 (A). To control for protein loading, parallel blots containing extracts from the same animals were probed with an antibody to ß-tubulin (A). Densitometry of the resulting blots (B) demonstrated a main effect of time postpartum (P < 0.0001) on both phospho- and total STAT5. Both phospho-STAT5 (black boxes) and total STAT5 (gray boxes) decreased on Day 37 postpartum versus Day 8. Each bar represents the LS mean ± SEM for 9 and 12 dams on Days 8 and 37 postpartum, respectively. Asterisks indicate statistical significance (P < 0.05) for the effect of day. There was no effect of day postpartum or genotype on ß-tubulin abundance

DISCUSSION

The administration of GH to lactating dairy cows has been demonstrated to enhance lactation persistence [37]. Although the actions of GH on milk synthesis have been hypothesized to involve the direct actions of IGF1 within the mammary gland [38], this has not been directly tested. The primary goal of these studies was to determine if the overexpression of IGF1 within the mammary glands of transgenic mice would enhance lactation persistence. Our data demonstrate that overexpression of des(1–3)IGF1 in the mammary gland supported greater rates of weight gain in cross-fostered litters during prolonged lactation. This observation, coupled with the fact that mammary gland wet weight was increased in the WAP-DES mice, supports the conclusion that milk synthesis was maintained for a longer period of time in these animals than in nontransgenic dams. The observation that milk composition was similar among the two genotypes also supports the conclusion that the loss of biosynthetic capacity in the mammary glands of WAP-DES mice was delayed and that enhanced growth of the litters could not have been attributed to altered milk nutrient content. Intriguingly, although loss of mammary secretory function was delayed in the WAP-DES dams, it was not entirely blocked. Thus, milk synthesis in the WAP-DES dams ultimately also became inadequate to supply the needs for weight gain in the cross-fostered pups despite the fact that mammary epithelial content was maintained to a greater extent. In addition, although mammary epithelial content was increased relative to nontransgenic dams, some loss of epithelial tissue still occurred in WAP-DES dams as well. These observations support the conclusion that IGF1 can delay the loss of milk yield during prolonged lactation in mice by slowing the loss of secretory cells. They further suggest that the actions of IGF1 in the lactating mammary gland may be limited to the regulation of cell turnover and of little consequence for milk synthesis.

Studies in both rodent models and dairy animals suggest that the ability of GH to support normal lactation is associated largely with effects on secretory cell number or mammary DNA content [39–41]. In lactating mice, mammary gland development defects due to prolactin receptor deficiency were corrected by administration of GH [40]. In lactating dairy cows, administration of GH increases the fraction of Ki67-positive cells, supporting the suggestion that secretory cell proliferation is increased [42]. In the current study, BrdU labeling tended to be higher in the WAP-DES mice, suggesting that cell proliferation might be increased in an analogous fashion to that observed in cattle. In addition, as was the case with the WAP-DES mice, administration of exogenous GH to dairy cows slows the rate at which milk yield declines during prolonged lactation but does not completely abolish this decline [23]. These observations suggest that factors beyond the direct actions of the GH:IGF1 axis also contribute to the loss of milk synthesis that occurs during prolonged lactation.

Because we had previously demonstrated that WAP-DES transgene expression is localized to the mammary gland during pregnancy and lactation, we reasoned that any effects on lactation would occur through direct local effects on IGF1 signaling within the mammary gland [25]. The changes that we observed in both body weight and composition in the WAP-DES mice, however, were consistent with the idea that expression of the transgene was also having an impact on other physiological processes in these animals. In our initial characterization of the WAP-DES mice, we measured serum samples collected at 10 days postpartum for human IGF1 but failed to detect any [25]. The fact the human IGF1 in the present experiment was undetectable in plasma from WAP-DES mice at Day 8 postpartum agrees with our earlier results. The presence of high plasma human IGF1 in the WAP-DES mice at Day 37, however, suggests that with prolonged lactation the transgene protein is either being actively secreted or leaking into the bloodstream. Leakage of des(1–3)IGF1 into circulation of the WAP-DES mice during prolonged lactation is the most plausible explanation for the elevated plasma values because previous studies have demonstrated that mammary epithelial tight junctions become more open during late lactation [43]. This opening of the tight junctions has been shown to be blocked by treatment with exogenous prolactin during late lactation in rabbits and can be induced in lactating rats by inhibiting prolactin secretion with bromocryptine [44, 45]. In addition, this opening of mammary-tight junctions may be a key mediator allowing for transgenically expressed proteins to reach potential sites of action either on the basolateral surfaces of the epithelium or at other organs in the body. This idea has been suggested as a means to explain observations in other mammary-specific transgene models of IGF action and suggests that there is a potential for secreted transgene proteins to have both local effects within the gland as well as systemic actions on the whole animal [46].

The systemic action of the WAP-DES transgene was evident not only from the increased plasma concentrations of human IGF1 but also from the decreased endogenous IGF1 concentration, the elevated blood and pituitary prolactin level, the increased body weight of the dams, and their altered body composition. The lowering of endogenous plasma IGF1 is consistent with feedback inhibition by IGF1 of GH secretion by the pituitary, which thereby reduces endogenous IGF1 production [47]. The perplexing part here, however, is that endogenous IGF1 was low both on Day 8, postpartum when human IGF1 was below the limit of detection in our assay, and on Day 37, when human IGF1 was elevated. A possible explanation for this is that because the des(1–3)IGF1 transgene protein does not interact with IGF binding proteins, it may act as free IGF1 in circulation [48, 49]. Thus, secretion of this protein into the bloodstream at levels too low to be detected by our assay may still have had some biological activity. This interpretation is particularly relevant in light of recent data suggesting that circulating levels of free IGF1 may be more important than total IGF1 levels in the regulation of GH secretion [50]. The fact that this IGF1 was biologically active at least some time before Day 37 is clearly illustrated by the effects on body weight and composition. The fact that lean body mass was increased in these mice is consistent with previous studies in which exogenously administered IGF1 enhanced nitrogen retention in normal female rats [51]. Part of this response, however, may have also been related to the fact that plasma prolactin was elevated and has the ability to stimulate food intake [52]. Previous work in wild-type and IGF1 knockout mice has demonstrated that IGF1 can stimulate prolactin secretion in nonlactating virgin mice [53]. Our data extend these observations by providing evidence in support of the idea that IGF1 may be an important regulator of prolactin secretion during lactation. These observations suggest that the effects of WAP-DES on lactation were mediated at least partially through indirect effects on the pituitary and on nutrient balance.

Data from a number of studies now support the idea that mammary cell turnover is an important determinant of the loss of milk synthesis during prolonged lactation. In both dairy cows and goats, significant amounts of apoptosis have been detected in the mammary gland as lactation progresses, and changes in mammary DNA content have been shown to accompany losses of milk synthesis during prolonged lactation [42]. Our own work has demonstrated that similar losses of mammary DNA occur in the mouse with prolonged lactation (unpublished observations). We have also observed that apoptosis increases in the mammary gland with prolonged lactation. In addition, with dams in which lactation was sustained to 20 days postpartum by a single cross-fostering of young pups at Day 17, apoptosis was significantly decreased in WAP-DES mice compared to their nontransgenic siblings [27]. Consequently, we expected that mammary cell apoptosis might be reduced in the WAP-DES mice on Day 37 postpartum as well. The fact that mammary epithelial content was higher in the WAP-DES mice than in control mice at Day 42 suggests that although apoptosis was not changed on the Day 37, it may have differed at some point earlier in lactation. On the other hand, the trend (P < 0.09) toward increased BrdU labeling in WAP-DES mice on Day 37 suggests that potential effects of the transgene on epithelial cell proliferation, rather than apoptosis, may have contributed to the increased epithelium found in the WAP-DES mice by the end of the study.

Overexpression of IGF1 within the mammary glands of lactating mice can decrease mammary cell apoptosis during involution [26, 32]. The observation that apoptosis increased in both groups of dams on Day 37 suggests that increasing cell death is a significant contributor to the overall loss of cells that occurred with prolonged lactation. However, the observation that the WAP-DES transgene failed to inhibit apoptosis on Day 37 postpartum suggests that overexpressed IGF1 may not have been able to activate cell survival pathways in the mammary gland during prolonged lactation. Our previous work [26] with WAP-DES mice has demonstrated that overexpression of des(1–3)IGF1 reduced apoptosis during natural but not forced involution on Day 20 postpartum. This differential effect was linked to the expression of the IGF-dependent signaling proteins, insulin receptor substrates (IRS) 1 and 2, the expression of which was lost with forced involution. In the present study, we observed a significant increase in the phosphorylation of IGF1Rß, supporting the conclusion that at least by Day 37 postpartum, the overexpressed des(1–3)IGF1 was capable of acting locally on the mammary epithelium. However, the fact that phosphorylation of IRS1 and AKT was not increased in the WAP-DES mice suggests that these pathways were not responsive to chronic stimulation by IGF1 during prolonged lactation and supports the suggestion that a potential block exists in the activation of this pathway upstream of IRS1.

Prolactin is a key hormone to normal mammary gland development and lactation, and the concentrations of this hormone in blood decrease with prolonged lactation [54, 55]. The observation that STAT5 phosphorylation decreased during prolonged lactation agreed with our finding that plasma prolactin was decreased and is consistent with reports in the literature. Although the finding that prolactin decreases in blood during prolonged lactation has been previously reported, the results presented in this study are the first to correlate this loss with decreased prolactin-dependent signaling capacity in the gland. Surprisingly, however, STAT5 phosphorylation, when expressed as a ratio to total STAT5, was only modestly elevated in the WAP-DES mice even though they had over a 2-fold increase in plasma concentrations of prolactin. The reason for this discrepancy at present is unclear but also could be explained by potential downregulation in the signaling pathway upstream of STAT5.

In summary, overexpression of IGF1 in the mammary gland is capable of enhancing milk synthesis for a finite period during prolonged lactation. This effect appears to be the result of presumably local actions as well as the indirect systemic effects that were observed. However, the observation that the WAP-DES transgene was incapable of sustaining increased milk production indefinitely suggests the involvement of other unknown factors that regulate both cell turnover and metabolic activity.

ACKNOWLEDGMENTS

The authors thank Mr. Gregory Shelton and Mr. Walter Olea for technical assistance with the analysis. Thanks also to Dr. Robert J. Collier for providing a critical editorial analysis of this manuscript. The authors would also like to thank Ms. Jeanette Schoppe for editorial assistance. The authors also thank Ms. Joanne Pratt and Dr. Ken Ellis of the CNRC Body Composition Core for help with the Piximus analysis.

FOOTNOTES

1 Supported by USDA NRI grant 2001–35206–11145 (D.L.H.) and by NIH grant DK52197 (D.L.H.), and NIH grant CA94118 (A.V.L.). This work is a publication of the United States Department of Agriculture/Agricultural Research Service Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine Texas Children's Hospital, Houston, Texas. The contents of this publication do not necessarily reflect the views or policies of the United States Department of Agriculture, nor does the mention of any trade names, commercial products, or organizations imply endorsement by the United States government. Back

2 Correspondence: Darryl L. Hadsell, USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, 1100 Bates St., Houston, TX 77030. FAX: 713 798 7057; dhadsell{at}bcm.tmc.edu Back

Received: 24 May 2005.

First decision: 15 June 2005.

Accepted: 2 August 2005.

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