Diminished Milk Synthesis in Upstream Stimulatory Factor 2 Null Mice Is Associated With Decreased Circulating Oxytocin and Decreased Mammary Gland Expression of Eukaryotic Initiation Factors 4E and 4G

Darryl L. Hadsell, Sharon Bonnette, Jessy George, Daniel Torres, Yann Klementidis, Shan Gao, Peter M. Haney, Joan Summy-Long, Melvyn S. Soloff, Albert F. Parlow, Mario Sirito and Michele Sawadogo

United States Department of Agriculture/Agricultural Research Service Children’s Nutrition Research Center (D.L.H., S.B., J.G., D.T., Y.K., S.G., P.M.H.), Department of Pediatrics and Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030; Department of Pharmacology (J.S.-L.), The Pennsylvania State University, The Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033; Department of Obstetrics and Gynecology (M.S.S.), University of Texas Medical Branch, Galveston, Texas 77555; Harbor-UCLA Medical Center (A.F.P.), Torrance, California 90509; and Department of Molecular Genetics (M.Si., M.Sa.), The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

Address all correspondence and requests for reprints to: Darryl Hadsell, United States Department of Agriculture/Agricultural Research Service Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030. E-mail: dhadsell{at}bcm.tmc.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Previous studies have suggested that upstream stimulatory factors (USFs) regulate genes involved with cell cycle progression. Because of the relationship of USFs to an important oncogene in breast cancer, c-myc, we chose to determine the importance of USF to normal mammary gland development in the mouse. Expression of USF in the mammary gland throughout development demonstrated only modest changes. Mutation of the Usf2 gene was associated with reduced fertility in females, but had no effect on prepartum mammary gland development. However, lactation performance in Usf2-/- females was only half of that observed in Usf2+/+ females, and both lactose and nitrogen were decreased in milk from Usf2-/- dams. This decrease was associated with diminished mammary tissue wet weight and luminal area by d 9 of lactation and with a decreased protein-DNA ratio. This decrease was associated with reduced abundance of the eukaryotic initiation factors eIF4E and eIF4G. Blood oxytocin concentrations on d 9 postpartum were also lower in Usf2-/- mice than Usf2+/+ mice. In contrast, the mutation had no effect on blood prolactin concentrations, mammary cell proliferation or apoptosis, mammary tissue oxytocin receptors, or milk protein gene expression. The mutation had only modest effects on maternal behavior. These data support the idea that USF is important to physiological processes necessary for the establishment and maintenance of normal lactation and suggest that USF-2 may impact lactation through both systemic and mammary cell-specific mechanisms.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
UPSTREAM STIMULATORY FACTOR (USF) consists of the basic helix-loop-helix (bHLH)/zipper proteins USF-1 and -2, which are related to the c-myc family of transcription factors (1). Characterization of the cDNAs for these proteins has revealed the existence of variant mRNAs for both USF-1 and -2 (1, 2, 3). Binding (3, 4) and in vitro transcription studies (4) have demonstrated that these proteins function as heterodimers to activate transcription through binding to E box elements (5) identical in sequence to those to which myc binds (6, 7). In addition, both USF and myc interact with TFII-I to form complexes at promoter initiator elements that activate or repress transcription, respectively (8, 9). Binding of myc has been shown to activate transcription of genes required for cell-cycle progression and/or cell growth [reviewed by Grandori et al. (10)]. Transcriptional activation by c-myc depends on its ability to heterodimerize with max (11). This activation is competitively inhibited by mad proteins such as mad1 (12) and mxi1 (13), which compete with myc for heterodimerization with max. The resulting inhibition would be expected to block myc-dependent cell cycle progression and has been suggested to promote differentiation (14). The ability of USF to compete with myc/max for the same DNA-binding site supports the idea that USF cooperates with the myc/max/mad network to regulate cellular function. The exact ramifications of this functional interaction between USF and myc, however, have been difficult to discern because of variations in USF action as a function of promoter context (15) and/or cellular context (16).

In mammary epithelial cells, myc is an important regulator of cell cycle progression. Transgenic- or retroviral-mediated overexpression of myc in the mouse mammary gland causes abnormal increases in mammary cell proliferation (17, 18). This phenotype has been observed in conjunction with a lactation defect. Despite these observations, little work has been reported on the functional significance of the myc/max/mad network to the processes governing normal mammary gland development and lactation. In addition the mechanism by which myc regulates mammary cell function and the importance of myc-related proteins, such as USF-1 and -2, to normal mammary gland development and lactation are poorly understood.

Mice that carry targeted mutations in the genes for USF-1 or -2 have been characterized. Targeted deletion of the Usf1 gene has little apparent effect on overall development (19, 20). In contrast, USF-2 null mice have been reported to exhibit decreased viability, growth retardation, and decreased lifespan (20, 21). During the course of initial studies on the Usf2 null mice, the offspring of female homozygous USF-2 null mice were observed to be nonviable. This lack of viability was believed to be caused by an inability of USF-2 null dams to lactate. Because of the known interaction of USF with the myc/max/mad family of proteins and because both myc and USF regulate expression of genes necessary for cell cycle progression, we hypothesized that USF-2 was necessary for mammary gland development and/or lactation. The goals of the present studies, therefore were 1) to measure the expression of USF-1 and -2 in the mammary gland throughout pregnancy and lactation; and 2) to determine the effects of a targeted germline mutation of the USF-2 gene on mammary gland development and function during pregnancy and lactation.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The mammary gland undergoes dramatic changes in both morphology and gene expression profiles throughout a single pregnancy-lactation cycle. To determine whether USF expression in the mammary gland relates to these changes, the developmental regulation of USF-1 and -2 protein and mRNA abundance was analyzed. To identify the predominant proteins present in mammary tissue extracts that bind USF/myc binding sites, EMSAs were conducted with nuclear extracts isolated from the mammary tissue of lactating mice. Incubation of these extracts with a 32P-labeled oligonucleotide containing the consensus USF/myc binding site (22) revealed the presence of a single, abundant, DNA-binding complex (Fig. 1AGo, lane 1). Preincubation of the binding reaction with antibodies specific for either USF-1 (Fig. 1AGo, lane 2) or USF-2 (Fig. 1AGo, lane 3) resulted in supershifted complexes that were not observed with either an antibody specific for c-myc (Fig. 1AGo, lane 4) or with normal rabbit serum (Fig. 1AGo, lane 5). Abundance of USF-1 and -2 proteins in mammary tissue extracts changed modestly over the course of a developmental cycle with both proteins showing 1.5- to 2-fold elevation (P < 0.05) at midpregnancy (Fig. 1Go, B and F). Both proteins were highest at d 16 of pregnancy and lowest at d 10 of lactation. Ribonuclease (RNase) protection analysis (RPA) used probes that were specific for exons 5–7 and 4–5 for USF-1 and -2, respectively (Fig. 1CGo). The combined use of a USF-1 and cyclophilin probe with mammary gland RNA produced six RNase-protected fragments (Fig. 1DGo). Fragment 1 migrated at the size expected for USF-1. Fragment 6 migrated at the size expected for cyclophilin. Hybridization of the individual USF-1 and cyclophilin probes (data not shown) to mammary RNA or yeast RNA suggested that fragments 2, 3, and 5 were also specific for the USF-1 mRNA while fragment 4 was a cyclophilin probe that was resistant to RNase digestion (see Fig. 1DGo, lane 8). Similarly, combined use of the USF-2 and cyclophilin probes resulted in multiple bands when hybridized to mammary RNA (Fig. 1EGo). Protected fragments 1 and 5 migrated at the size predicted for the USF-2 and cyclophilin mRNAs, respectively. Likewise, hybridization of the individual USF-2 probe to mammary RNA suggested that fragments 2 and 4 were also USF-2 specific while fragment 3 is the same RNase-resistant fraction of the cyclophilin probe observed in the USF-1 RPA (Fig. 1EGo). To quantitate abundance of USF mRNAs, densitometric data were obtained for the sum of either USF-1-specific, USF-2-specific, or cyclophilin-specific bands (Fig. 1GGo). These analyses demonstrated that USF mRNAs exhibited modest changes over the course of gestation and lactation. For USF-1, the changes in mRNA abundance appeared to parallel those observed for cyclophilin, the internal control. The largest change for USF-1 occurred with mammary involution and had little relationship to the abundance of USF-1 protein. For USF-2, significant, but small depressions in mRNA were observed during late pregnancy and lactation. This change was similar to that observed for cyclophilin, which displayed small increases during early pregnancy and then declined with late pregnancy and lactation. Overall, the protein and RNA data support the conclusion that relatively small changes occur in USF-1 and -2 expression within the mammary gland over the course of a single pregnancy and lactation cycle.



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Fig. 1. USF Protein and mRNA Abundance Varies Little During Mammary Gland Development

DNA binding activity was detected by EMSA on 2 µg of nuclear extract prepared from lactating mammary tissue (A). A control reaction contained only nuclear extract, and probe (lane 1). Supershift reactions were conducted by preincubating the nuclear extracts with antibodies to USF-1 (lane 2), USF-2 (lane 3) c-myc (lane 4), or normal rabbit serum (lane 5). Western blotting was used to measure the abundance of USF-1 and -2 in mammary homogenates (B). Samples were analyzed from virgin, 8-d, 16-d, or 18-d pregnant, 10-d lactating, or 3-d involuting mice (panel B, lanes 1–6, respectively). The blots represent three independent replications. Developmental regulation of USF-1 and -2 mRNA abundance was measured by RPA. A diagram of the USF-1 and -2 mRNAs illustrates the location and relative sizes of the USF probes (C). Total RNA from mammary tissue was hybridized to antisense riboprobes for either USF-1 (D) or USF-2 (E). Samples were analyzed from virgin, 8-d, 16-d, or 18-d pregnant, 10-d lactating, or 3-d involuting mice (panel B, lanes 2–7, respectively). After hybridization, the reactions were digested with RNase A and T1. These were electrophoresed alongside a 100-nucleotide (range 100–500 nucleotides) size marker (lane 1), a yeast RNA-negative control (lane 8), and the undigested probes (lane 9). An antisense probe for cyclophilin was used as an internal control in all reactions. The expected size of the protected fragments was 370, 249, and 103 nucleotides for USF-1, -2, and cyclophilin, respectively. Densitometry data for the Western blot and RPA analysis are shown in panels F and G, respectively. Each bar is the mean ± SEM for two to four mice. Bars with superscripts highlight statistically significant differences (P < 0.05). a, Significantly greater than V, 18P, 10L, and 3I. b, Significantly greater than V, 8P, 18P, 10L, and 3I. c, Significantly greater than 18P and 10L. d, Significantly greater than V and 10L.

 
To determine the importance of USF to development of the mammary gland, studies were done on previously described mice that carry a targeted mutation of the Usf2 gene (20). Initially, expression of USF-1 and -2 within the mammary glands of these Usf2-/- mice was characterized. Western blotting for both USF-1 and -2 proteins in mammary gland extracts prepared from mice at d 1 postpartum demonstrated decreased abundance in the Usf2+/- mice as compared with Usf2+/+ mice (Fig. 2Go A). This decrease was even more dramatic in Usf2-/- mice whereas the abundance of ß-actin in these same extracts showed only minor variation. The loss of USF-2 protein in mammary tissue of the Usf2-/- genotype was also evident in frozen tissue sections stained for USF-2 and E-cadherin (Fig. 3CGo). Immunofluorescent staining of mammary gland sections from Usf2+/+ mice demonstrated the presence of USF-2 protein within the nuclei epithelial cells during both pregnancy and lactation (Fig. 3Go, A and B). No USF-2, however, was visible in the Usf2-/- mammary tissue. This decrease in USF-2 protein correlated with the results of RNase protection assays that demonstrated a reduction in the mRNA for USF-2 (Fig. 2CGo), but not USF-1 (Fig. 2BGo) in the mammary tissue from Usf2+/- and Usf2-/- mice. Thus mutation of the Usf2 gene deceased both the mRNA and protein for USF-2. In contrast, USF-1 expression was affected by the mutation only at the level of protein abundance.



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Fig. 2. Comparison of USF-1 and -2 Protein and mRNA Abundance in Mammary Tissue from Lactating Wild-Type and USF-2 Null Mice

Western blots (A) were used to measure USF-1 and -2 protein abundance. Tissue extracts were prepared from mammary gland of (+/+) (lanes 1 and 2), (+/-) (lanes 3 and 4), and (-/-) (lanes 5 and 6) at 1 d postpartum. Thirty micrograms of protein were loaded in each lane. The blots were reprobed for ß-actin as a loading control. RPAs for USF-1 (B) or USF-2 (C) were conducted on total RNA isolated from mammary tissue of (+/+) (lanes 1 and 2), (+/-) (lanes 3 and 4), or (-/-) (lanes 5 and 6) mice. The data presented in this figure are representative of five animals for each genotype.

 


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Fig. 3. Nuclear Localization of USF-2 Mammary Epithelial Cells During Pregnancy and Lactation

Frozen sections of mammary tissue were prepared from Usf2+/+ mice at 16 d of pregnancy (A) or 10 d of lactation (B) or Usf2-/- mice at 3 d of lactation. The sections were stained for E-cadherin (red) to identify epithelial cells, and USF-2 (green). Strong nuclear localization of USF-2 is obvious from the pattern of staining in USF2+/+ tissue both during pregnancy and lactation. USF-2 staining is dramatically decreased in the Usf2-/- mammary tissue (C). Each image is representative of at least three mice. Scale bar, 100 µm.

 
The effect of disrupting the Usf2 gene on mammary gland development in virgin mice was determined by analysis of whole mounts prepared from 8-wk-old Usf2+/+ and Usf2-/- mice (Fig. 4Go, A and B, respectively). Overall comparison of the whole mounts revealed that in both genotypes, mammary epithelium occupied the entire area of the mammary fat pad. Comparison of epithelial area in tagged image file format (TIFF) images collected from randomly sampled fields of tissue within each whole mount demonstrated that the percentage of epithelium was similar among mammary glands from Usf2+/+ and Usf2-/- mice (34.7 ± 1.5 and 35.7 ± 2.5%, respectively). Using a similar approach, mammary gland development at d 8 of pregnancy was also compared between Usf2+/+ and Usf2-/- mice. This comparison revealed no obvious differences between the two genotypes at this time point (Fig. 4Go, C and D). In addition, the percent epithelium within mammary whole mounts was similar for both Usf2+/+ and Usf2-/- (43.3 ± 3.9 and 48.0 ± 2.9%, respectively).



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Fig. 4. Mammary Ductal Development Is Normal in Virgin and Early Pregnant Usf-/- Mice

Mammary gland whole mounts were prepared from 8-wk-old virgin (A and B) and 8-d pregnant (C and D), Usf2+/+ (A and C), and Usf2-/- (B and D) female mice. Extent of epithelial development was measured in bitmap images collected from mice of both genotypes. A and B represent four and seven images of each Usf2+/+ and Usf2-/- mouse, respectively. C and D represent five and three images of each Usf2+/+ and Usf2-/- mouse. In glands from virgin mice, the percent epithelial area was 34.7 ± 1.5 and 35.7 ± 2.5% for Usf2+/+ and Usf2-/- mice, respectively. In glands from 8-d pregnant mice, the percent epithelial area was 43.3 ± 3.9 and 48.0 ± 2.9% for Usf2+/+ and Usf2-/- mice, respectively. Scale bar, 2 mm.

 
To identify functional effects during reproduction that relate to the loss of USF-2, cohorts of female Usf2+/+ and Usf2-/- littermates were induced to ovulate and then mated to FVB males. During the course of these studies, Usf2-/- females were observed to have lower (P < 0.001) conception rates than their Usf2+/+ siblings (34% and 71%, respectively). Moreover, litter size in Usf2-/- mice was half (P < 0.05) of that observed in Usf2+/+ mice (5 ± 1 and 10 ± 1 pups per litter, respectively), and individual pup viability at birth was also decreased (P < 0.0001) (47% and 99%, respectively). In virgin animals of both genotypes, however, ovarian morphology was similar (data not shown) as were blood concentrations of gonadal steroid hormones (P > 0.05) (Usf2+/+ vs. Usf2-/-; estrogen (E), 24.6 ± 9.5 vs. 26.9 ± 8.8 pg/ml; progesterone (P), 18.4 ± 8.4 vs. 7.6 ± 2.9 ng/ml). Parturition, however, was delayed (P < 0.0001) in the Usf2-/- mice as compared with Usf2+/+ mice (20.7 ± 0.2 and 19.0 ± 0.1 d, respectively). Despite these reproductive defects, Usf2-/- females could lactate if placed with litters of foster pups. Comparison of the average body weights of Usf2+/+ and Usf2-/- dams on d 1 postpartum demonstrated that the loss of USF-2 causes decreased (P < 0.0001) body weight (29.4 ± 0.7 and 22.0 ± 0.5 g, respectively). The ability of Usf2+/+ and Usf2-/- females to lactate was analyzed by measuring the growth of cross-fostered pups over the first 8 d of lactation (Fig. 5AGo). The growth of pups suckled on Usf2-/- dams was only 39% (P < 0.01) of that for pups suckled on Usf2+/+ dams, indicating that either maternal behavior and/or volume or nutrient content of the milk were insufficient to support normal growth of the litter.



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Fig. 5. Loss of USF-2 Decreases Lactation Performance

In panel A, a random mixture of (+/+) and (+/-) pups were cross-fostered onto Usf2+/+ (+/+) or Usf2-/- (-/-) USF-2 mice, and litter weight was measured over the first 8 d of lactation. On d 1 postpartum, each dam was placed with a litter of 12 pups. These litters consisted of a random mixture of (+/-) and (+/+) pups that were 1 d old. Average pup weight was measured by weighing the entire litter and then dividing by the number of pups in the litter. Each dam was milked on d 9 postpartum. Milk volume is shown in panel B. Each symbol represents the mean ± SEM for seven and five dams of (+/+) and (-/-) genotype, respectively.

 
To determine whether milk composition was altered in Usf2-/- mice, milk samples were collected on d 8 postpartum and analyzed for lactose, water, fat, and nitrogen (Table 1Go). The total volume of milk collected was less (P < 0.05) for Usf2-/- dams than that recovered from Usf2+/+ dams (Fig. 5Go B). Milk water and fat were similar in the two genotypes whereas both milk lactose and milk nitrogen content were lowered (P < 0.05) in Usf2-/- mice as compared with Usf2+/+ mice (Table 1Go). For milk lactose, the difference between Usf2-/- and Usf2+/+ was 83% whereas total milk nitrogen differed by 17%. These data suggest that the diminished capacity of Usf2-/- dams to rear their pups was associated with decreased milk volume as well as alterations in milk composition.


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Table 1. Comparison of Milk Composition in Usf2+/+ and Usf2-/- Mice

 
To determine the extent to which the lactation defect in the Usf2-/- mice was accounted for by decreases in expression of genes for milk proteins, the abundance of the mRNAs for several major milk proteins, as well as for genes involved with lactose biosynthesis, was measured. Northern blot analysis of RNA extracted from mammary tissue obtained from mice at d 1, d 3, and d 9 post partum demonstrated that the abundance of mRNAs for ß-casein, {alpha}-lactalbumin, and whey acidic protein (WAP) were similar, regardless of genotype (Fig. 6AGo). Similarly, using a multiprobe RNase protection assay no differences were observed in the abundance of mRNAs for ß-casein, {alpha}-lactalbumin, ß-galactosyl transferase, or glucose transporter 1 (GLUT1) at 9 d postpartum (Fig. 6Go, C and D). Likewise, Western blot analysis (Fig. 6Go B) using a milk-specific antibody demonstrated that the abundance of the major milk proteins in mammary tissue at 9 d postpartum was similar among Usf2-/- and Usf2+/+ mice. These results suggest that mutation of the Usf2 gene does not interfere with expression of specific milk proteins or with genes important to lactose synthesis.



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Fig. 6. Milk Protein Gene Expression Is Little Affected by Loss of USF-2

Abundance of milk protein mRNAs was analyzed by Northern blotting (A) on d 1 (lanes 1–4), d 3 (lanes 5–12), and d 9 (lanes 13–20) postpartum and by multiprobe RPA (C) on d 9 postpartum. For the Northern blot, 10 µg total RNA were loaded per lane. The resulting blot was then probed with 32P-labeled cDNA probes for {alpha}-lactalbumin ({alpha}-Lac), WAP, and ß-casein (ß-Cas). The ethidium bromide-stained gel (EthBr) is shown to confirm RNA loading. For the RPA, 20 µg of total RNA was hybridized to a riboprobe mixture containing specific probes for ß-galactosyl transferase, {alpha}-lac, ß-Cas, and GLUT1. Specificity of the probes is demonstrated in panel C. A single protected fragment is detected for each of the probes when hybridized to mammary RNA (lane 3). Hybridization to yeast RNA (lane 2) fails to produce protected fragments. Abundance of milk proteins in mammary tissue was analyzed by Western blotting (B) of extracts prepared from mammary tissue isolated at 9 d postpartum from Usf2+/+ (lanes 1, 2, 5, and 6) and Usf2-/- (lanes 3, 4, 8, and 9) mice. Mammary tissue extracts were loaded at 10 ng/lane (B). Loading was confirmed in parallel Coomassie-stained gels. Densitometric analysis of the autoradiographs generated from the RPA assay demonstrated no change in the mRNAs in response to the Usf2 mutation (D). Each bar represents the means ± SEM for eight and four Usf2+/+ (+/+) and Usf2-/- (-/-) mice, respectively.

 
Because effects of the Usf2 mutation on the mammary gland do not materialize until the onset of lactation and because the Usf2-/- mice attain a smaller body size than their Usf2+/+ siblings, it is possible that the lactation defect is due to postpartum endocrine defects. During our initial study on pup growth, a blood sample was collected from both Usf2+/+ and Usf2-/- females on d 9 postpartum by cardiac venipuncture after avertin anesthesia. Based on the analysis of these samples, blood oxytocin (OT) was found to be 48% lower (P < 0.03) in Usf2-/- than Usf2+/+ mice. The secretion of OT and its circulating concentration, however, is known to vary with the suckling stimulus and anesthetics that alter activation of the magnocellular neuroendocrine system (23). For these reasons, we did a second study in which the collection of trunk blood occurred immediately after the milk ejection reflex displayed by the litter. This behavioral response occurs in synchrony with the bolus release of OT and its hormonal effect causing milk ejection to the pups (24). This response is easily observed in conscious free-roaming rats and is known to be associated with indices of milk ejection (25). Pups nursing Usf2-/- and Usf2+/+ dams were observed for a stretch response on d 3, d 6, and d 9 postpartum. The stretch response was imperceptible in pups younger than 6 d. However on d 6 and d 9 this behavior of the litter was obvious. Under these experimental conditions (experiment 2), circulating concentrations of OT in response to suckling on d 9 postpartum were found to be higher (P < 0.02) than those observed in our first study (experiment 1), where samples were collected from anesthetized dams with no regard for the timing of sample collections. (Fig. 7AGo). This difference in hormone concentrations between experiments also could have been related to the assays used to measure OT or to differences in the method of sample collection (i.e. cardiac puncture under anesthesia vs. decapitation of conscious dams). Importantly, the difference in blood OT between Usf2+/+ and Usf2-/- dams was similar in both experiments. Analysis of the pooled data from both experiments demonstrated that blood OT was 54% higher (P < 0.03) in Usf2+/+ than in Usf2-/- mice (Fig. 7BGo). Prolactin (PRL) was also measured in blood samples collected on d 9 at the time of a stretch response. Blood PRL was similar in Usf2+/+ and Usf2-/- dams (Fig. 8BGo). These data suggest that the reduced lactation performance in Usf2-/- mice was associated with modestly reduced blood OT, but not PRL.



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Fig. 7. Decreased Lactation Performance in Usf2-/- Mice Is Associated with Diminished Blood OT Levels and Minimal Effects on Mammary OT Receptors, Myepithelial Cell Number and Area, and the Stretch Response of the Litter

Blood OT was compared among Usf2+/+ and Usf2-/- dams in two separate experiments (A). In the first (EXP1) blood was collected by cardiac puncture from anesthetized dams. In the second experiment (EXP2) trunk blood was collected after decapitation of conscious dams immediately after the third stretch reflex of the litter. Analysis of the pooled results from both experiments demonstrated that blood OT was significantly reduced in Usf2-/- dams (B). Receptors for OT were measured by binding assays on microsomes isolated from the mammary gland (C). Each data point represents the binding to a pooled microsome preparation prepared from three to five mice per genotype. Mammary myoepithelial cells were detected by immunostaining (D) for {alpha}-smooth muscle actin (green) while total cell nuclei were detected by TOPRO-3 staining. The scale bar indicated 100 µm. The percentage of myoepithelial cells and average myoepithelial cells area was determined by automated analysis of the green- and red-stained particles in each of 10 fields from each specimen using image-Pro Plus (E). Lastly, evidence of milk ejection was obtained by determining the frequency of stretch responses in the suckling pups over the first 15 min of nursing (F). Asterisks indicate significant differences (P < 0.05).

 


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Fig. 8. Maternal Behavior Is Only Modestly Impacted, and Blood PRL Is Unaffected by Mutation of the Usf2 Gene

Latency of pup retrieval was measured on d 1, d 3, and d 6 postpartum over a 15-min period after a 5-min separation period (A). Animals that failed to retrieve their pups in the 15-min period were assigned a latency of 15. Each bar represents the median latency for 5 Usf2+/+ and Usf2-/- mice. On d 9 postpartum, trunk blood samples were collected after decapitation of conscious dams immediately after the third stretch reflex of the litter (B). Asterisks indicate a significant difference (P < 0.05). Each bar represents the mean ± SEM for a minimum of five animals per genotype.

 
To determine whether mammary gland responsiveness to OT was reduced in Usf2-/- mice, receptor binding studies were carried out on mammary membranes using 125I-labeled OT antagonist. The binding capacity for the OT antagonist was found to be similar mammary membranes from Usf2+/+ and Usf2-/- mice (Fig. 7CGo). Both the number and size of mammary myoepithelial cells, as measured by Immunofluorescence for {alpha}-smooth muscle, were also similar among Usf2+/+ and Usf2-/- mice (Fig. 7Go, D and E). Lastly, the frequency of stretch responses in the suckling pups, monitored as an indicator of effectual OT secretion/milk ejection, was similar among Usf2+/+ and Usf2-/- dams (Fig. 7Go F). These data suggest that the mammary glands in Usf2-/- mice are capable of responding to OT and that the milk ejection reflex occurred in the Usf2-/- mice in response to suckling.

Lactation defects have been associated with abnormal maternal behavior in a number of mutant mouse stains (26, 27, 28). Consequently, we chose to compare maternal behavior among Usf2+/+ and Usf2-/- dams during early lactation. Normal maternal behavior in lactating mice includes nest building, retrieving and grouping of pups in the nest, anogenital licking, and crouching over the pups in the nest to provide warmth and nursing. Several of these behaviors are known to depend on PRL (26). Although casual observation of the Usf2-/- dams during lactation did not reveal any obvious behavioral defects, we chose to determine whether pup retrieval was impaired in the Usf2-/- dams. Latency of pup retrieval was measured on d 1, d 3, and d 6 postpartum. To initiate the observations, both the dam and the pups were removed from the cage and weighed. The dam was then immediately replaced in the cage. At 5 min after their removal from the cage, the pups were placed back into the cage at the end opposite the nest. Retrieval times were then recorded for up to 15 min following the replacement of the pups. On d 1 postpartum, the median retrieval latency was 2-fold higher (P < 0.04) in Usf2-/- dams compared with their Usf2+/+ siblings (Fig. 8AGo). By d 3, this difference had diminished and on d 6, retrieval latencies were similar among Usf2+/+ and Usf2-/- dams. These observations suggest that defects in maternal behavior contributed only modestly to the lactation defect.

To determine the extent to which diminished lactational capacity in the Usf2-/- mice was explained by alterations in mammary gland development, tissue morphology, mammary gland wet weight, DNA, and protein were compared. Analysis of hematoxylin-eosin-stained mammary tissue sections from mice at 9 d postpartum demonstrated a modest decrease in the ratio of epithelium to stroma (Fig. 9EGo) and a dramatic decrease in luminal area (Fig. 9FGo) for Usf2-/- mice (Fig. 9DGo) as compared with those of Usf2+/+ mice (Fig. 9CGo). These differences, however, were not apparent on d 1 postpartum (Fig. 9Go, A and B). These data correlated with the lower volume of milk recovered from Usf2-/- mice. Wet weight (Fig. 9GGo) of the inguinal (no. 4) mammary glands was also modestly less (P < 0.01) in Usf2-/- mice than that of Usf2+/+ mice. This decreased weight coincided with increased (P < 0.001) mammary tissue DNA concentration (Fig. 9HGo), decreased total mammary gland protein (P < 0.05), and a decreased ratio of protein to DNA (P < 0.001) (Fig. 9IGo). In addition, the mammary glands of Usf2+/+ and Usf2-/- mice had similar rates of bromodeoxyuridine (BrdU) incorporation (5.5 ± 0.5 and 5.2 ± 4.0%, respectively) and apoptosis (0.4 ± 0.2 and 0.6 ± 0.3, respectively) on d 3 postpartum, indicating that the lactation defect in the Usf2-/- mice was due to diminished biosynthetic capacity rather than alterations in mammary gland development.



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Fig. 9. Decreased Lactation in Usf2-/- Mice Is Associated with Decreased Mammary Wet Weight, Epithelial Area Alveolar Luminal Area, and Decreased Total Protein

Hematoxylin-eosin-stained mammary tissue sections were evaluated from Usf2+/+ (A and C) and Usf2-/- (B and D) mice on d 1 (A and B) and d 9 (C and D) postpartum. The scale bar indicates 200 µm. Percent epithelial area (E) and alveolar luminal area (F) was measured in TIFF images captured from 10 random fields for each sample. The bars represent the means ± SEM for five to seven mice. Mammary wet weight (G), DNA and protein concentration (H), DNA and protein mass and protein-DNA ratio (I) were measured on the no. 4 mammary glands on d 9 postpartum. Bars with different superscripts differ (P < 0.05). Asterisks indicate significant effects of genotype. *, P < 0.001; #, P < 0.05.

 
A major action of myc is to regulate cell growth (29). A number of studies have revealed that myc regulates the transcription of genes important for translation and ribosome biogenesis (15, 30). A few of these genes have also been demonstrated to be USF responsive at least in cell culture models (15, 31). To determine whether loss of USF-2 could affect factors that influence translation, we measured the expression of two eukaryotic initiation factors in mammary tissue eIF4E and eIF4G. Western blotting of mammary tissue extracts prepared from mice at either 3 d or 9 d postpartum demonstrated the presence of a single band (~25 kDa for eIF4E) and as many as three bands between 97 and 220 kDa for eIF4G (Fig. 10Go, A and B). This banding pattern is consistent with previous data on eIF4G (32, 33, 34). Densitometric analysis of the Western blots revealed that the abundance of eIF4E was significantly reduced (P < 0.05) in mammary tissue taken at d 9 postpartum compared with d 3 (Fig. 10Go C). In addition, both eIF4E and eIF4G were significantly less (P < 0.05) in mammary tissue from Usf2-/- mice than that from Usf2+/+ mice (Fig. 10Go D). Because there was no interaction between day postpartum and genotype, only the main effects are presented. The abundance of eIF4E and eIF4G in mammary tissue from Usf2+/+ mice was 114% and 67% higher, respectively, than that in mammary tissue from Usf2-/- mice. These changes appear to be the result of a posttranscriptional or posttranslational effect as RT-PCR analysis of total mammary gland RNA demonstrated that abundance of the transcripts for these two proteins was similar among Usf2+/+ and Usf2-/- mice (data not shown). These results strengthen the conclusion that USF-2 functions in lactating mammary cells to support translation and that the diminished milk synthesis observed in Usf2-/- mice was at least partially due to reduced mammary gland protein synthesis.



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Fig. 10. Abundance of Translation Initiation Factors eIF4E and eIF4G Is Decreased in Usf2-/- Mice

Tissue extracts were prepared from mammary glands of Usf2+/+ (+/+) and Usf2-/- (-/-) mice on d 3 (A) and d 9 (B) postpartum. The extracts were then electrophoresed (30 µg/lane) on SDS-PAGE gels and then transferred to polyvinylidenedifluoride membrane. The resulting blots were then probed with antibodies specific for either eIF4G or eIF4E. Equal loading among the samples was controlled for by analysis of parallel gels stained with Coomassie (data not shown). Densitometry data were then analyzed as a two-factor experiment (day x genotype). This analysis demonstrated a statistically significant (P < 0.05) effect (*) due to day on eIF4E abundance (C) and a significant effect due to genotype on both eIF4E and eIF4G (D). The interaction was not significant. Each bar represents the mean ± SEM for four to five animals.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Our results demonstrate that targeted inactivation of the USF-2 gene in mice reduces both body weight and fertility in females and prolongs gestation in those females capable of establishing pregnancy. The mutation of the Usf2 gene also causes a mild, but readily detectable, impairment of lactation. This impairment is associated with decreased milk volume coincident with a reduced alveolar lumen area and with decreases in both milk lactose and milk nitrogen. In addition, DNA concentration increased, while total protein, the ratio of protein to DNA, and the abundance of eIF4E and eIF4G in the mammary glands from Usf2-/- mice decreased. These alterations in mammary gland function occurred only during the postpartum period and were associated with modestly decreased blood concentrations of OT in response to suckling. No changes were observed in blood PRL, mammary OT receptors, myoepithelial cell number, or frequency of OT release as indicated by the stretch reflex of the litter. Furthermore, loss of USF-2 had no impact on mammary cell proliferation or differentiation, and only minor impact on maternal behavior. Initially, we believed that the actions of USF proteins within the mammary gland might be explained in terms of their relationship to c-myc. However, the observations that we have collected from the Usf2-/- mice provide more support for the conclusion that the actions of USF in the mammary gland reach beyond that which occurs through a direct competition with c-myc for DNA binding sites.

The bHLH proteins are an evolutionarily conserved family of transcription factors that can be divided into five subgroupings on the basis of DNA binding preferences and amino acid sequence (35). Their actions have been described to be of importance in myogenesis, neurogenesis, sex determination, and cell lineage determination where these are most frequently attributed to regulation of cell proliferation or differentiation (35). The USF proteins, along with c-myc and other members of the myc/max/mad network, belong to the most ancient subgroup of the bHLH family, subgroup B. Numerous studies have been published on the biological significance of interactions among these proteins. The Mad proteins function as antagonists to myc proteins through their ability to compete in the formation of heterodimers with max (12, 36). The mechanism through which gene activation or repression occurs in response to this competition is now believed to be due to modifications in histone acetylation in response to the recruitment of either transformation/transcription domain-associated protein (TRRAP) or Sin3 by myc or mad, respectively (37, 38, 39). In contrast, the USF proteins do not bind directly to members of the myc/max/mad network, but rather interact with the same DNA response elements as myc (40, 41). The significance of this competition from a transcriptional standpoint, however, is complex as the ability of USF to supplant or antagonize myc is dependent on the specific promotor and cell type involved (16). Moreover, the effects of mutating the Usf2 gene may not involve myc since USF proteins are also recognized to not interact with the same proteins as myc (42). Thus, further studies are needed to clarify the mechanism of USF action in the mammary gland in relationship to those of myc.

Although the role of the myc/max/mad network in cancers has been extensively studied and a number of studies have been published on the interactions of myc and USF at the level of gene transcription, the role of these proteins in normal developmental processes is poorly understood. To our knowledge there have been no published studies on the necessity of these proteins to normal mammary gland development or to lactation. Our analysis of USF expression in the mammary gland suggests that these genes are only minimally affected by the hormonal changes that occur during a pregnancy-lactation cycle and that the dramatic changes in proliferation and differentiation of mammary cells during this cycle do not require marked alterations in USF-1 or -2 protein levels. Similarly, only small changes in the abundance of USF-1 and -2 mRNAs in the mammary gland during the course of a pregnancy-lactation cycle can be seen in the recently published microarray data by Master et al. (http://www.abramsoninstitute.org/chodoshdata.html) (43). On the other hand, previously published analysis of myc gene expression in the mammary gland has demonstrated that this gene is highly expressed during times of elevated mammary cell proliferation and decreased with mammary cell differentiation during lactation (44).

The use of genetically engineered mouse models to understand the actions of bHLH proteins in mammary gland development and lactation have, at present, been of limited use. Targeted inactivation of the genes for c-myc and N-myc causes embryonic death, precluding an analysis of myc action in the mammary gland (45, 46, 47). Overexpression of myc in the mammary gland causes a hyperproliferative defect in the mammary epithelium that is associated with impaired cellular differentiation, impaired lactation, and the formation of mammary adenocarcinomas (18, 48). Mice carrying mutations of both the Usf1 and Usf2 genes exhibit early embryonic lethality (20), suggesting some analogy with the phenotype observed in myc mutants. Targeted mutation of the Usf1 gene alone, however, produces little observable phenotypic change (19, 20). In contrast, targeted mutation of the Usf2 gene decreased USF-1 expression, resulting in an overall decrease in total USF levels. In addition, Usf2-/- mice have been shown to have altered expression of hepatic genes involved with gluconeogenesis and lipogenesis, diminished pre- and postnatal growth, and reduced fertility in males (20, 21, 49). Our analysis of USF expression in mammary glands of Usf2-/- mice also demonstrated decreased abundance of USF-1 protein, but not mRNA. A high degree of evolutionary conservation among the untranslated regions has been observed for the USF mRNAs (3). Therefore, the regulation of USF-1 expression at the translational or posttranslational level is not unreasonable. Both this result and the observation that Usf2-/- mice are smaller than Usf2+/+ mice are consistent with previous observations in this model (20).

Although the underlying mechanism through which mutation of the Usf2 gene affects reproduction is currently unclear, our preliminary results suggest that despite statistically significant decreases in conception rates and litter sizes, this defect is relatively subtle and not associated with dramatic changes in ovarian morphology or in blood concentrations of E and P. Normal mammary ductal development in the virgin mouse is known to depend on adequate circulating concentrations of E and P and mutation of estrogen receptor and progesterone receptor causes dramatic reductions in mammary ductal and alveolar development, respectively (50, 51). Our finding that mammary development was similar in Usf2+/+ and Usf2-/- females both before and during pregnancy agrees with the observation that circulating concentrations of E and P were similar. These data, coupled with our observations on mammary gland morphology and BrdU labeling during lactation, suggest that USF-2 is not important to normal mammary gland development.

The mechanism through which mutation of Usf2 affects lactation is currently unclear. In mice, pup growth is the most frequently used indicator of milk yield and functional indicator of successful lactation. Although this is a crude measurement it correlates well with other types of measurements used to assess lactation such as 3H2O turnover in the pups (52). The pup growth data, combined with our observations on milk volume recovered after administration of exogenous OT, and on milk composition, support the conclusion that the decreased growth of pups fostered to the Usf2-/- dams is due to both diminished milk synthesis and altered milk composition. Our data on mammary gland wet weight, morphology, and DNA and protein are also consistent with the conclusion that the synthesis of milk is reduced in the mammary glands of Usf2-/- mice. The causes of this decreased milk synthesis, however, are likely multifaceted and involve both systemic and mammary cell autonomous mechanisms.

The observation that body weight is reduced in Usf2-/- mice supports the possibility that the lactation deficiency may have been partially due to a systemic mechanism rather than solely a mammary cell autonomous mechanism. In fact, genetic selection for milk yield in both mice and cattle has been associated with increase in body weight as well as increased mammary gland wet weight and DNA content (53, 54). If mammary development had been abnormal in Usf2-/- mice either during the early postnatal period or during pregnancy, then mammary tissue transplantation would have been a useful technique to rule out systemic effects. However, because the defect was specific to the postpartum period, only a tissue-specific conditional deletion of the Usf2 gene within the mammary gland would have been useful for discerning between systemic and cell autonomous effects. In the absence of such a model, we examined the mice for changes in PRL and OT, systemic hormones necessary for lactation.

Because milk protein gene expression was normal in the Usf2-/- mice, we were not surprised to find that the mutation had no effect on blood PRL concentrations. Previous studies have shown that haploinsufficiency for the PRL receptor causes a lactation defect characterized by both decreased expression of the milk protein genes and reduced mammary gland development (55). Maternal behavior has also been demonstrated to be severely impaired in mice with haploinsufficiency for the PRL receptor (26). Behavior in the Usf2-/- females was only modestly affected. Collectively, these observations suggest that defective PRL signaling did not contribute to the diminished milk synthesis observed in Usf2-/- mice.

OT is also known to play an essential role in lactation. OT null mice are incapable of lactation (56). This defect is associated during the early postpartum period with decreased mammary cell proliferation and increased mammary cell death resulting in premature involution of the gland (57). Although the decreased circulating levels of OT observed in Usf2-/- mice represents an attractive explanation for the lactation defect, neither proliferation nor apoptosis of mammary cells was altered in Usf2-/- mice, arguing against this possibility. In addition, the absence of changes in the number of observable stretch reflexes within the litters suggests that, despite the decreased blood concentration, similar numbers of milk-ejection episodes occurred in response to the suckling stimulus. Although blood levels of OT were adequate to elicit a stretch response, the duration and/or intensity of milk ejection may still have been reduced in the knockout animals. Studies have shown that intensity of the stretch response increases with increasing doses of exogenous OT (24). However, the connection between these types of changes and altered milk composition in the Usf2-/- mice is unclear.

At present, there appears to be little relationship between blood OT and milk composition. In dairy cows, long-term treatment with exogenous OT can increase milk synthesis without altering milk composition (58). However, to our knowledge, these types of studies have not been done in rodent models. The reduction in lactose concentrations within the milk from Usf2-/- mice was somewhat surprising in that most species do not show marked variations in milk lactose concentration. In rodents, however, this is not entirely unexpected as milk lactose concentration varies over the course of lactation and is sensitive to malnutrition (59, 60). In studies where lactose has been directly manipulated in mice through genetic approaches, these alterations have been shown to directly affect both the volume and water content of milk (61, 62, 63). In this regard, if decreased lactose were the sole cause of the diminished milk synthesis, then increased concentrations of both fat and nitrogen would have been expected in the milk from Usf2-/- mice. Rather, the combined decreases in milk lactose and nitrogen, coupled with an overall decrease in total mammary gland protein content and in the ratio of protein to DNA, suggests that more generalized inhibition of protein synthesis resulted in the reduced synthesis of milk in Usf2-/- mice. The fact that milk protein gene expression remains unchanged in Usf2-/- mice also supports the conclusion that the lactation defect is due to reduction in overall protein synthesis.

In addition to involvement in cell cycle regulation, c-myc regulates cell growth (64). In this respect, myc had been shown to regulate the transcription of genes involved with metabolism such as ornithine decarboxylase (65), genes involved with translation initiation such as eIF4E, eIF2{alpha}, and eIF4G, and genes important to ribosome assembly, such as nucleolin (15, 30). Consequently, it was encouraging to observe that the abundance of the eIF4E and eIF4G proteins was reduced in mammary tissue from the Usf2-/- mice. The surprising part of these observations, however, was that the mRNAs for these two proteins were unchanged between Usf2+/+ and Usf2-/- mice. This observation provides further support for the role of USF in supporting mammary cell protein synthesis during lactation. However, the fact that the changes in eIF4E and eIF4G were only observed at the protein level suggests again that the mechanism of USF action in the mammary gland is distinct from, and may even be independent of, interactions with c-myc. Future experiments that focus on further analysis of the OT system in Usf2-/- mice and that use mammary gland-specific conditional mutations of the Usf2 gene will be necessary to fully clarify the cell biology underlying the lactation defect.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Animals
All animal procedures were conducted in accord with the National Institute of Health Guidelines for Care and Use of Experimental Animals. Female FVB mice (Charles River Laboratories, Wilmington, MA) were killed at various times during mammary gland development during pregnancy and lactation. The developmental times studied were 6-wk-old virgins; 8, 16, and 18 d of pregnancy; 10 d of lactation; and 3 d of mammary involution after weaning at 10 d of lactation. For each developmental time point, mammary tissue was collected from three mice. The tissue was snap frozen in liquid nitrogen and then stored at -70 C until homogenized and assayed. Mice carrying a targeted disruption of the Usf2 gene have been previously described (20). For these experiments, the mutation was carried on a hybrid background consisting of 50% CD-1 and 50% FVB. To prevent confounding due to genetic background, all comparisons were made among littermates. Mammary tissue samples collected from virgin mice at 8 wk of age were used to compare mammary ductal development in whole mounts of the tissue. To initiate analysis of lactation, cohorts of (+/+) and (-/-) siblings were induced to ovulate by ip injection with 2.5 IU of Gestyl (Professional Compounding Centers of America Inc., Houston, TX) followed 47 h later by 2.5 IU of Pregnyl (Organon, Inc., West Orange, NJ) and then mated to FVB males. A total of four separate experiments were conducted. In the first study, mice were weighed and then euthanized on d 1 postpartum. In the second experiment, mice were placed with litters that consisted of a random mixture of 10 Usf2+/+ and Usf2+/- pups. Pup growth rates served as a measurement of the lactation performance for each dam. Average weights of the dam and pups were then measured for each litter from d 1 through d 8 postpartum. On d 8 postpartum a milk sample was obtained and on d 9 postpartum, the dams were weighed and euthanized under avertin anesthesia to provide mammary tissue samples and blood samples. Blood samples in this experiment were collected by cardiac puncture. In the third experiment, the mice were placed with cross-fostered litters as in experiment 2. Each dam was labeled with BrdU (100 mg/kg body weight) on d 3 postpartum and then euthanized. In the fourth experiment, mice were placed with cross-fostered litters and observed on d 1, 3, 6, and 9 for defects in maternal behavior. On d 1, 3, and 6, pup retrieval latencies were measured as follows: pups were separated from the dam for a period of 5 min and then returned to the cage opposite the nest; the time required for the retrieval of all 10 pups in the litter was monitored for 15 min. Dams that failed to retrieve their pups were assigned a latency of 15 min (900 sec). On d 3, 6, and 9, the dams were separated from their litters for 6 h. After this, their pups were returned and the stretch responses of the litter were monitored for 15 min. The stretch response is a classic behavior of the suckling pups that is associated with milk ejection mediated by a bolus release of OT from the magnocellular neuroendocrine system (24). On d 9 postpartum, trunk blood was collected from the dams after decapitation immediately after the third stretch response.

EMSA
Nuclear extracts were prepared as previously described (66). The synthetic double-stranded oligonucleotide used in these studies was a previously described 23 mer (22) containing a USF-1 consensus 5'-cac ccg gtc acg tgg cct aca cc-3'. The oligonucleotides were labeled with 32P as previously described (67). The labeled probes were gel purified on a 15% nondenaturing polyacrylamide gel. EMSAs were performed as described previously (68). Supershift analysis was performed using polyclonal antibodies to USF-1, USF-2, and c-myc (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Histology/Immunostaining
Whole mounts of the inguinal (no. 4) mammary gland were fixed in Tellyesniczky’s fixative and stained with iron-hematoxylin as previously described (69). Grayscale TIFF images of each whole mount were collected at a magnification of x12.5 using a Olympus SZ40 stereo zoom microscope (Olympus Corp., Lake Success, NY) fitted with a Dage CCD72. The percentage of epithelium occupying the whole mount was then measured in subsamples of each image using the image analysis software, Scion Image (Scion Corp., Frederick, MD). The morphology of lactating mammary tissue was visualized in hematoxylin-eosin-stained sections of paraffin-embedded tissue fixed overnight in 10% neutral-buffered formalin. To provide mammary gland samples for proliferation analysis, Usf2+/+ and Usf2-/- mice were labeled with BrdU (Sigma Chemical Co., St. Louis, MO) for 2 h at d 3 postpartum. Anti-BrdU immunohistochemistry was used to detect labeled mammary cells, while the TdT-mediated dUTP nick-end labeling assay was used to measure cell death. These procedures were conducted as previously described (70, 71). The percentage of cells proliferating or dying was quantified by counting at least 1000 cells per section. Expression patterns of USF-2 were analyzed by incubating frozen sections of mammary tissue fixed in 4% paraformaldehyde with a 1:8000 dilution of anti-USF-2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4 C. Alexafluor 488-conjugated antirabbit (Molecular Probes Inc., Eugene, OR) was used at 1:1000 dilution as a second antibody. Rat anti-E-cadherin was use at a dilution of 1:1000 (Zymed Laboratories, South San Francisco, CA) in combination with Alexafluor 633 to specifically stain epithelial cells. A fluorescein isothiocyanate-conjugated anti-{alpha}-smooth muscle actin (Sigma Chemical Co.) was used at 1:250 dilution to stain myoepithelial cells and TOPRO-3 (Molecular Probes Inc.) was used at 0.5 µM as a nuclear counterstain. Stained sections were visualized using the Fluoview FV300 Confocal Microscope (Olympus America Inc., Melville, NY). Myoepithelial cell numbers were estimated by automated counting of cells positive for {alpha}-smooth muscle actin. Total cell counts were estimated by automated counting of TOPRO3-stained cell nuclei. Counts were collected from TIFF images collected from 10 fields per specimen using Image-pro Plus image analysis software (Media Cybernetics, Silver Spring, MD).

RNA Analysis
Total RNA was isolated from mammary tissue using TRIZOL (Life Technologies Inc., Gaithersburg, MD) according to the manufacturer’s instructions. The mouse cDNAs for USF-1 and USF-2 have been previously described (1, 3). Templates for riboprobes were constructed by cloning PCR products generated from the cDNAs. The USF-1 template was constructed by designing primers that would amplify a product that spanned exons 4–7 (Fig. 1Go C). The primer oligos used to amplify this segment of the USF-1 cDNA were as follows: USF-1F 5'-gta cgt ctt ccg aac tga ga-3' and USF-1R 5'-tca cca caa gaa gta ttg cag-3'. The USF-1 primers generated a 370-bp PCR product that was then cloned directly into a pBSK-T-vector constructed according to Ref. 72 . The USF-2 template was constructed by amplifying a region that spanned exons 4 and 5 of the USF-2 cDNA. The primer oligos used to amplify this region were as follows: USF-2F 5'-tca gcg tcg tgt cca ccg-3' and USF-2R 5'-tgg gcg ata cca cag ctg tgt-3'. The USF-2 primers generated a 249-bp product that was cloned as described for the USF-1 template. Both the USF-1 and USF-2 cloned products were confirmed by sequencing. Templates for antisense probes were prepared by digesting the plasmids with NotI enzyme and subsequent purification and concentration using the Geneclean kit according to the manufacturer’s protocol (ISC Bioexpress, Kaysville, UT). Sense probes were generated by digesting with XhoI. Templates were used to make 32P-labeled riboprobes by in vitro transcription using the Promega Riboprobe Combination T7/T3 system (Promega Corp., Madison, WI). Gel-purified riboprobes for USF-1, -2, and cyclophilin were then used in RPA assays performed with the RPA II kit from Ambion (Ambion, Inc., Austin, TX). Hybridization reactions conducted with mouse mammary gland RNA produced major protected fragments of 370, 249, and 103 nucleotides for USF-1, -2, and cyclophilin, respectively. Hybridization reactions conducted with yeast RNA or a sense USF probes (data not shown) failed to yield RNase-protected fragments. RPA for ß-1,4-galactosyltransferase, {alpha}-lactalbumin, ß-casein, and GLUT1 was carried out separately from the previously described RPAs and yielded protected fragments of 430, 319, 250, and 187 nucleotides, respectively. These assays were performed as described above except that riboprobes were labeled nonisotopically using the BrightStar Biodetect Kit (Ambion, Inc.). Northern blotting was conducted with total mammary gland RNA using previously described cDNA probes for {alpha}-lactalbumin, ß-casein, and WAP (73, 74, 75).

DNA/Protein Analysis/Western Blotting
Mammary tissue DNA content was analyzed using the procedure of Labarca and Paigen (76). Mammary tissue protein content was determined in total homogenates using the BCA protein assay (Pierce, Rockford, IL). Total tissue protein extracts were prepared by homogenizing 50 mg of tissue in 250 ml of a lysis buffer (71). The extracts were electrophoresed on 12% SDS-PAGE minigels. Each lane was loaded with 30 µg extract protein. Equal loading was confirmed in parallel gels, which were stained with Coomassie blue. Proteins were transferred onto nitrocellulose (Schleicher & Scheull, Keene, NH) overnight at 45 mAmp in the presence of Towbin buffer (25 mM Tris, 192 mM glycine, 20% methanol). Blots were blocked for 4 h in Tris-buffered saline (TBS) (20 mM Tris, pH 7.5; 150 mM NaCl) plus 3% nonfat dry milk (Carnation, Glendale, CA). The membranes were incubated overnight with either rabbit USF-1 or rabbit anti-USF-2. Blots were then washed with TBS-T (20 mM Tris, pH 7.5; 150 mM NaCl, 0.1% Tween 20) three times (15 min once and 5 min twice), with agitation. The membranes were then washed three times in TBS containing 0.1% Tween 20 and incubated with horseradish peroxidase-conjugated goat antirabbit (Amersham Pharmacia Biotech, Arlington Heights, IL) (1:1000 dilution) for 30 min at room temperature. The blot was then washed four times for 15 min each and then developed using the enhanced chemiluminescent detection system (Pierce Chemical Co.) following the manufacturer’s directions. Blots were reprobed with an antibody against ß-actin (Sigma Chemical Co.) as a loading control. Milk protein abundance was measured by Western blot using an antibody specific to mouse milk proteins (Accurate Chemical & Scientific Corp., Westbury, NY). Individual caseins and WAP were identified on the basis of reported electrophoretic mobilities (77). Abundance of translation factors was also measured using Western blotting with rabbit polyclonal antibodies (1:1000) specific for eIF4E and eIF4G (all from Santa Cruz Biotechnology, Inc.). Equal sample loading within individual lanes was ensured by analysis of parallel gels stained with Coomassie blue.

Milk Analysis
In the second lactation experiment, milk was collected on d 9 postpartum under gentle vacuum from anesthetized dams after an injection of OT (1.5 U im in each hind leg). The milk was then flash frozen in liquid nitrogen before analysis. For each sample, the volume and the content of water, fat, nitrogen, and lactose were measured. The percentage of water (% wt/wt) was measured as weight loss after drying. Milk fat (% wt/wt) was measured using a modification of the procedure of Jeejeebhoy et al. (78). Milk nitrogen (% wt/wt) was measured using a Kjeldhal-phenol-hypochlorite technique (79). Milk lactose was determined using an YSI 2700 Select analyzer (YSI, Inc., Yellow Springs, OH). The composition of milk was then compared with previously published characteristics of murine milk (80).

Hormone Measurements
RIA for mouse PRL was performed by 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.1 Estrogen and progesterone were measured using ELISA kits (DSL Laboratories, Webster, TX). Blood OT was measured by two separate assays. In experiment 1, the measurements were made using a specific ELISA (Assay Designs Inc., Ann Arbor, MI). For experiment 2, blood OT was measured using a previously described RIA (81). OT receptor expression was measured using a previously described binding assay on mammary gland microsomal membranes (82).

Data Analysis
All of the quantitative data were analyzed by one-way or two-way ANOVA using SPSS. The number of animals used for each genotype ranged from three to seven. Differences were tested for with a significance level of {alpha} = 0.05. The pup weight data for each individual dam were used to calculate a daily growth rate by simple linear regression. Conception rates were compared using the {chi}2 test. Pup retrieval data were analyzed using the Mann-Whitney U test.


    ACKNOWLEDGMENTS
 
The authors thank Ms. Stella Tsang, Ms. Tatiana Alexeenko, and Ms. Vuong Bui for technical assistance. Thanks also to Dr. Joanne Richards for help in evaluating the ovaries in Usf2(-/-) mice.


    FOOTNOTES
 
This work was supported by federal funds from the United States Department of Agriculture/Agricultural Research Service under cooperative agreement no. 58-6250-6001 (to D.L.H.) and by Grant CA79578 (to M.S.).

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 and 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 trade names, commercial products, or organizations imply endorsement by the United States Government.

1 Consult web site, http://www.humc.edu/hormones. Send E-mail to parlow{at}humc.edu or fax (310) 222-3432 for additional information. Back

Abbreviations: bHLH, Basic helix-loop-helix; BrdU, bromodeoxyuridine; E, estrogen; GLUT, glucose transporter; OT, oxytocin; P, progesterone; PRL, prolactin; RNase, ribonuclease; RPA, RNase protection analysis; TBS, Tris-buffered saline; USF, upstream stimulatory factor; WAP, whey acidic protein.

Received for publication January 22, 2002. Accepted for publication July 30, 2003.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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