Intramammary infusion of leptin decreases proliferation of mammary epithelial cells in prepubertal heifers

High energy intake and excessive body fatness impair mammogenesis in prepubertal ruminants. High energy intake and excessive fatness also increase serum leptin. Our objective was to determine if an infusion of leptin decreases proliferation of mammary epithelial cells of prepubertal heifers in vivo. Ovine leptin at 100 μg/quarter per d with or without 10 μg of insulin-like growth factor (IGF)-I was infused via the teat canal into mammary glands of prepubertal dairy heifers; contralateral quarters were used as controls. After 7 d of treatment, bromodeoxyuridine was infused intravenously and heifers were slaughtered ∼2 h later. Tissue from 3 regions of the mammary parenchyma was collected and immunostained for bromodeoxyuridine (BrdU), proliferating cell nuclear antigen (Ki-67), and caspase-3. Leptin decreased the number of mammary epithelial cells in the S-phase of the cell cycle by 48% in IGF-I-treated quarters and by 19% in saline-treated quarters. Leptin did not alter the number of mammary epithelial cells within the cell cycle, as indicated by Ki-67 labeling. Caspase-3 immunostaining within the mammary parenchyma was very low in these heifers, but leptin significantly increased labeling in saline-treated quarters. Leptin enhanced SOCS-3 expression in IGF-I-treated quarters but did not alter SOCS-1 or SOCS-5 expression. We conclude that a high concentration of leptin in the bovine mammary gland reduces proliferation of mammary epithelial cells. The reduced proliferation is accompanied by an increase in SOCS-3 expression, suggesting a possible mechanism for leptin inhibition of IGF-I action. Whether leptin might be a physiological regulator of mammogenesis remains to be determined.     

Feeding an enhanced diet to Holstein heifers during the preweaning period alters steroid receptor expression and increases cellular proliferation

Preweaning diet and estradiol treatment alters mammary development. Our objectives were to study the effects of diet and estradiol on proliferation of mammary epithelial cells and expression of estrogen receptor α (ESR1) and progesterone receptors (PGR) in these cells. Thirty-six Holstein heifer calves were raised on (1) a control milk replacer fed at 0.44 kg of powder/head per day, dry matter (DM) basis (restricted, R; 20.9% crude protein, 19.8% fat, DM basis), or (2) an enhanced milk replacer fed at 1.08 kg of powder/head per day, DM basis (Enhanced, EH; 28.9% crude protein, 26.2% fat, DM basis). Milk replacer was fed for 8 wk. At weaning, a subset (n = 6/diet) of calves were euthanized and had tissue harvested. Remaining calves received estradiol implants (E₂) or placebo and were euthanized at wk 10 to harvest tissue. Treatments were (1) R, (2) R + E₂ (R-E2), (3) EH, and (4) EH + E₂ (EH-E2). One day before euthanasia calves were given bromo-2′-deoxyuridine (BrdU; 5 mg/kg of body weight). At euthanization, mammary parenchyma was removed and fixed. Tissue sections from zone 1 (cisternal), 2 (medial), and 3 (distal) within the mammary gland were stained with hematoxylin and eosin and antibodies to measure expression of ESR1, PGR, and incorporation of BrdU. At wk 8, R-fed calves had more PGR-expressing cells in distal parenchyma; however, PGR expression intensity was greater in EH-fed calves. The proportion of cells expressing ESR1 was not affected by diet, but expression intensity (receptors per positive cell) was greater in EH-fed calves across all zones (62–81%). Overall, the percent BrdU-positive epithelial cells was 2 and 0.5 fold greater for EH-fed calves in zone 2 and 3. The proportion of labeled cells was greater in terminal ductal units than in subtending ducts, and treatment effects were more evident in terminal ductal units. At wk 10, calves treated with estradiol had 3.9-fold greater PGR expression intensity. The intensity and percent of cells expressing ESR1 was lowest in estradiol-treated calves. Overall, estradiol-treated calves had the greatest number of proliferating epithelial cells. Moreover, in zone 3, EH-E2 calves had a higher percentage of proliferating cells than in all other treatments. Results indicate both diet and estradiol administration alter proliferation rates of the mammary epithelium and that changes in expression of ESR1 and PGR are involved in enhanced mammary development. The data support our hypothesis that enhanced preweaning feeding increases the mammary tissue responsiveness to mammogenic stimulation.     

Effect of estrogen on leptin and expression of leptin receptor transcripts in prepubertal dairy heifers

Plasma leptin concentrations increase as growing dairy heifers approach puberty and have greater plasma estrogen. In intact and ovariectomized rodents, estrogen has been shown to modulate expression of leptin and its receptor (Ob-R). To determine if estrogen regulates the bovine leptin system, prepubertal dairy heifers were ovariectomized at 140 d of age or left intact. A month later, both groups received a subcutaneous injection of excipient or 17β-estradiol for 3 consecutive days. Neither ovarian status nor 17β-estradiol injection altered plasma leptin or leptin mRNA abundance in adipose tissue depots. To assess whether these factors affected Ob-R expression, we tested 20 bovine tissues for leptin receptor (Ob-R) by using quantitative real-time PCR assays for the short receptor isoform (Ob-Ra), the long receptor isoform (Ob-Rb), and all receptor isoforms (Ob-R_TOTAL). Ob-R_TOTAL was detected in all tissues, with copy numbers covering 3 orders of magnitude between the lowest and highest expressing tissues (kidney cortex vs. liver). The Ob-Rb isoform accounted for 40% of Ob-R_TOTAL in the hypothalamus, but averaged less than 3% of Ob-R_TOTAL in peripheral tissues. Reciprocally, Ob-Ra accounted for only 19% of Ob-R_TOTAL in the hypothalamus and for nearly all of Ob-R_TOTAL in most peripheral tissues. Finally, we evaluated the effects of ovarian status and 17β-estradiol on Ob-R expression in selected tissues. Treatment with 17β-estradiol reduced Ob-R_TOTAL, Ob-Rb, and Ob-Ra expression by 70% in the uterine endometrium and tended to do the same in mammary adipose tissue. There was no effect of 17β-estradiol on Ob-R in the hypothalamus, liver, soleus muscle, or subcutaneous adipose tissue. We conclude that greater estrogen secretion does not cause increased plasma leptin in prepubertal dairy heifers but estradiol can modulate Ob-R expression in some estrogen-responsive tissues.     

Short communication: intramammary infusion of IGF-I increases bromodeoxyuridine labeling in mammary epithelial cells of prepubertal heifers

When dairy heifers are fed to gain more than 900 g of body weight/d, they have less mammary parenchymal DNA at puberty but more insulin-like growth factor-I (IGF-I) in serum. This negative relationship between serum IGF-I concentration and mammary epithelial cell proliferation is in disagreement with the extensively reported role of IGF-I as a stimulator of mammary epithelial cell proliferation. Despite the large body of evidence suggesting that an increase in IGF-I concentration should lead to an increase in mammary epithelial cell proliferation of prepubertal heifers, it had not been previously tested. Our objective was to determine if intramammary infusions of IGF-I would stimulate mammogenesis in prepubertal heifers in vivo. After 7 d of treatment, bromodeoxyuridine was infused intravenously and heifers were slaughtered 3 h later. Samples from 3 regions of the mammary parenchyma were collected, fixed, sliced, and incubated with bromodeoxyuridine monoclonal antibody to identify cells in the S-phase of the cell cycle. Intramammary infusion of IGF-I increased the percentage of epithelial cells in the S-phase by 52% (6.4 vs. 4.2%, ± 0.3%). Proliferation was similar in all 3 parenchymal regions, and the response to IGF-I was similar in each region. We conclude that local IGF-I increases proliferation of mammary parenchymal epithelial cells in prepubertal heifers.     

Partial feed restriction decreases growth hormone receptor 1A mRNA expression in postpartum dairy cows

Uncoupling of the growth hormone (GH) axis in early postpartum dairy cows is correlated with a decrease in liver GH receptor (GHR) 1A mRNA and a decrease in liver GH receptor protein. Postpartum recoupling of the GH axis is also correlated with GHR 1A mRNA and GHR protein. We hypothesized that dry matter intake (DMI) partially controls the increase in GHR 1A mRNA postpartum. Prepartum Holstein dairy cows (n = 11) were offered feed ad libitum. After calving, 6 cows were fed 70% of their expected DMI (feed restriction) for 14 d and 5 cows were fed ad libitum (control). Both groups were fed ad libitum after d 14. Liver was biopsied prepartum and on d 1, 7, 14, and 21 postpartum; blood was sampled throughout the experimental period. Rate of increase in postpartum milk production was less for feed-restricted cows. The GHR 1A mRNA decreased from prepartum to d 1 postpartum and subsequently increased. Rate of postpartum increase in GHR 1A mRNA was less in feed-restricted cows. Diminished GHR 1A persisted for at least 7 d after feed-restricted cows returned to ad libitum feeding. Liver insulin-like growth factor-I mRNA concentrations decreased from prepartum to d 1 as well, but were similar for feed restricted and control thereafter. We concluded that DMI partially controls GHR 1A mRNA expression in early postpartum dairy cows and that the decrease in GHR 1A in response to feed restriction persisted for at least 1 wk after ad libitum feeding was restored.     

Cellular proliferation rate and insulin-like growth factor binding protein (IGFBP)-2 and IGFBP-3 and estradiol receptor alpha expression in the mammary gland of dairy heifers naturally infected with gastrointestinal nematodes during development

Mammary ductal morphogenesis during prepuberty occurs mainly in response to insulin-like growth factor-1 (IGF-1) and estradiol stimulation. Dairy heifers infected with gastrointestinal nematodes have reduced IGF-1 levels, accompanied by reduced growth rate, delayed puberty onset, and lower parenchyma-stroma relationship in their mammary glands. Immunohistochemical studies were undertaken to determine variations in cell division rate, IGF-1 system components, and estradiol receptors (ESR) during peripubertal development in the mammary glands of antiparasitic-treated and untreated Holstein heifers naturally infected with gastrointestinal nematodes. Mammary biopsies were taken at 20, 30, 40, and 70 wk of age. Proliferating cell nuclear antigen immunolabeling, evident in nuclei, tended to be higher in the parenchyma of the glands from treated heifers than in those from untreated. Insulin-like growth factor binding proteins (IGFBP) type 2 and type 3 immunolabeling was cytoplasmic and was evident in stroma and parenchyma. The IGFBP2-labeled area was lower in treated than in untreated heifers. In the treated group, a maximal expression of this protein was seen at 40 wk of age, whereas in the untreated group the labeling remained constant. No differences were observed for IGFBP3 between treatment groups or during development. Immunolabeling for α ESR (ESR1) was evident in parenchymal nuclei and was higher in treated than in untreated heifers. In the treated group, ESR1 peaked at 30 wk of age and then decreased. These results demonstrate that the parasite burden in young heifers negatively influence mammary gland development, affecting cell division rate and parameters related to estradiol and IGF-1 signaling in the gland.     

Mammary localization and abundance of laminin, fibronectin, and collagen IV proteins in prepubertal heifers

The objective was to determine localization and abundance of extracellular matrix proteins fibronectin, laminin, and collagen in mammary tissues from ovariectomized or intact prepubertal heifers. Mammary parenchyma and fat pad tissues were collected from 14 6-mo-old heifers: eight were ovariectomized between 1 to 3 mo of age, and six were used as intact controls. Distribution of total collagen was assessed by Sirius Red staining of tissue sections. Fibronectin, laminin, and type IV collagen were assessed by immunohistochemistry. Abundance of fibronectin and laminin was also analyzed by western blotting. Total mammary mass was much less in ovariectomized animals (130 +/- 21 vs. 304 +/- 25 g). Histological structure differed as parenchyma from intact animals contained abundant, complex branching epithelial terminal ductular units, whereas terminal ductular units from ovariectomized animals were mostly major ductal structures with little or no branching. Collagen fibers were abundant and densely packed throughout interlobular stroma and were less abundant and more diffuse within intralobular stroma. Type IV collagen was primarily in basal lamina of mature ducts, whereas fibronectin and laminin staining were present throughout parenchymal stroma, in both intact and ovariectomized animals. Using western blotting, fibronectin was more abundant within parenchyma than in the fat pad and significantly higher in parenchyma from ovariectomized heifers. Laminin was more abundant in parenchyma from intact than ovariectomized animals (30 vs. 17 densitometric units/mg of tissue), but laminin was similar between parenchyma and fat pad. These results provide initial evidence that fibronectin, laminin, and collagen participate in regulation of heifer prepubertal mammary development.     

Effects of feeding prepubertal heifers a high-energy diet for three, six, or twelve weeks on mammary growth and composition

The experimental objective was to determine the effects of feeding prepubertal dairy heifers a high-energy diet for 3, 6, or 12 wk on mammary growth and composition. Holstein heifers (age = 11 wk; body weight = 107 ± 1 kg) were assigned to 1 of 4 treatments (n = 16/ treatment). The treatment period lasted 12 wk and treatments were H0 (low-energy diet fed for 12 wk, with no weeks on the high-energy diet); H3 (low-energy diet fed for 9 wk, followed by the high-energy diet for 3 wk); H6 (low-energy diet fed for 6 wk, followed by the high-energy diet for 6 wk); and H12 (high-energy diet for all 12 wk). The low- and high-energy diets were formulated to achieve 0.6 and 1.2 kg of average daily gain, respectively. Heifers were slaughtered at 23 wk of age and mammary tissue was collected. A longer duration of feeding the high-energy diet increased total mass of the mammary gland, extraparenchymal fat, and intraparenchymal fat, but did not alter the mass of fat-free parenchymal tissue. When adjusted for carcass weight to reflect differences in physical maturity, the mass of fat-free parenchymal tissue decreased in a linear fashion with a longer duration on the high-energy diet. Total masses of mammary parenchymal DNA and RNA were not different. However, after adjustment for carcass weight, the masses of DNA and RNA decreased as heifers were fed the high-energy diet for a longer duration. The percentages of epithelium, stroma, and lumen, the number of epithelial structures, and the developmental scores of mammary parenchymal tissue were not different among treatments. However, the percentage of proliferating epithelial cells in the terminal ductal units, as indicated by Ki-67 labeling, decreased as heifers were fed the high-energy diet for a longer duration. We concluded that feeding prepubertal heifers a high-energy diet for a longer duration resulted in a linear decrease in both the percentage of mammary epithelial cells that were proliferating and in the mass of fat-free mammary parenchyma per unit of carcass. High-energy feeding hastens puberty and, in this study, decreased mammary epithelial cell proliferation in areas of active ductal expansion. These data are consistent with the idea that feeding heifers a high-energy diet will reduce mammary parenchymal mass at puberty.     

Effect of bovine somatotropin and rumen-undegradable protein on mammary growth of prepubertal dairy heifers and subsequent milk production

Rapid body growth during the prepubertal period may be associated with reductions in mammary parenchymal growth and subsequent milk yield. The objective of this study was to test effects of dietary rumen-undegradable protein (RUP) and administration of recombinant bovine somatotropin (bST) during the prepubertal period on mammary growth and milk yield of dairy heifers. Seventy-two Holstein heifers were used in the experiment. At 90 d of age, 8 heifers were slaughtered before initiation of treatment. Remaining heifers were assigned randomly to 1 of 4 treatments. Treatments consisted of a control diet (5.9% RUP, 14.9% CP, DM basis) or RUP-supplemented diet (control diet plus 2% added RUP) with or without 0.1 mg of bST/kg of BW per day applied in a 2 x 2 factorial design. A total of 6 heifers per treatment (3 each at 5 and 10 mo of age) were slaughtered for mammary tissue analysis. Remaining heifers were bred to evaluate impact of treatment on subsequent milk yield and composition. Mammary parenchymal growth was not affected by RUP or bST treatment. Total parenchymal mass increased from 16 to 364 g, and parenchymal DNA from 58 to 1022 mg from 3 to 10 mo of age, respectively. Furthermore, number of mammary epithelial cells likely was not affected by diet or bST because the epithelial cell proliferation index, assessed by Ki-67 labeling, was not affected by treatment, nor was total parenchymal DNA and lipid content. Neither deleterious effects of increased rates of gain nor positive effects of bST were evident in prepubertal mammary growth. Subsequent milk production and composition was not different among treatments.     

Bovine somatotropin and rumen-undegradable protein effects on skeletal growth in prepubertal dairy heifers

The objectives of this study were to determine the effects of dietary rumen-undegradable protein (RUP) and bovine somatotropin (bST) during the period from weaning until puberty on body weight (BW) and skeletal growth rates and age at puberty. Fifty-one Holstein heifers at 90 d of age were randomly assigned to 4 treatment groups consisting of 0.1 mg/kg BW per day of bST and 2% added dietary RUP (dry matter basis) applied in a 2 x 2 factorial design (n = 13 per group, except bST with no RUP group, n = 12). From 90 to 314 d, bST increased average daily gain (ADG) by 0.07 kg/d and BW by 16.2 kg, while added RUP increased ADG by 0.10 kg/d and BW by 21.4 kg. Both bST and added RUP effects on BW and ADG were additive. Skeletal growth rates, as measured by withers height (WH) and hip height (HH) were increased by both bST and added RUP. Somatotropin and RUP increased WH by 1.8 and 2.7 cm and hip height by 2.5 and 4.0 cm, respectively, at 314 d of age. Growth curves showed that added RUP effects on rates of BW, WH, and HH growth were greatest from 90 to 150 d age and diminished thereafter, suggesting that protein was limiting during this time period. Conversely, bST effects tended to be greater as the heifers approached puberty, but only in the presence of added RUP. Age at puberty was not affected by treatment, averaging 314 d of age across treatments. From 314 to 644 d of age, rates of BW, WH, and HH growth were similar among treatment groups. However, treatment differences present at 314 d of age persisted through 644 d of age, more than 10 mo after treatments ceased. These results suggest that protein during the early postweaning period and bST during the 200 to 300 d of age period just prior to puberty could be used to accelerate simultaneous increases in both BW and skeletal growth rates in dairy heifers without reducing age at puberty.     

Effect of a biological response modifier on expression of growth factors and cellular proliferation at drying off

Agents that increase natural protective mechanisms have been proposed for the prevention and treatment of intramammary infections. Staphylococcus aureus is a major pathogen causing primarily subclinical chronic mastitis that responds poorly to antibiotic therapy. The objectives of this study were to describe the effects of a single intramammary infusion of a lipopolysaccharide-based biological response modifier (BRM) on mammary epithelial cellular proliferation and expression of insulin-like growth factor-I (IGF-I) and vascular endothelial growth factor (VEGF) in uninfected and Staph. aureus-infected bovine mammary glands during involution. Three groups of 12 cows, 6 Staph. aureus-infected and 6 uninfected, were infused with BRM or placebo in 2 mammary quarters and killed at 7, 14, and 21 d of involution. The proportion of infected quarters, mammary cell proliferation, and IGF-I and VEGF expression were evaluated. Biological response modifier treatment decreased the proportion of Staph. aureus-infected mammary quarters at 7 d of involution, but a similar number of isolations were observed at 14 and 21 d of involution in either treated or control quarters. The percentage of proliferating mammary epithelial cells was higher in infected than uninfected quarters at every observation period, irrespective of the treatment administered, whereas uninfected BRM-treated quarters showed increased cell proliferation at 7 d of involution. Insulin-like growth factor-I expression in uninfected quarters was not affected by treatment and showed a decrease at 21 d of involution. Expression of IGF-I was greater in infected than uninfected quarters at every observation period, irrespective of the treatment received. Expression of VEGF was greater in BRM-treated uninfected quarters at 7 d of involution compared with controls. In infected quarters, VEGF expression was lowest in BRM-treated quarters at 7 d of involution and increased throughout the observation period. Conversely, untreated infected quarters showed the highest VEGF expression at 7 d and decreased at 21 d of involution. Mammary cell proliferation and expression of IGF-I and VEGF were increased in Staph. aureus-infected quarters. Increased mammary cell proliferation and VEGF expression were observed in BRM-treated quarters during the first week of involution.     

Reduced insulin-like growth factor-I after acute feed restriction in lactating dairy cows is independent of changes in growth hormone receptor 1A mRNA

Growth hormone receptor (GHR) and insulin-like growth factor-I (IGF-I) mRNA decrease in the liver of dairy cows at parturition. Epinephrine is released and feed intake is decreased at the same time. The objective of this study was to determine whether feed restriction and (or) administration of epinephrine could recapitulate the changes in the hepatic GHR 1A and IGF-I mRNA that occur at parturition. Eight lactating cows were randomly assigned to one of two rations (ad libitum or restricted). The cows assigned to the restricted ration were fed 75, 60, 60, and 25% of feed consumed by the ad libitum cows on successive days to mimic the changes in feed intake around parturition. Liver samples were collected by needle biopsy before and after feed restriction. Cows received either 0.02 mg/kg of epinephrine or saline approximately 24 h before the second liver biopsy so that a 2 x 2 factorial arrangement of treatments was created. Feed restriction increased plasma nonesterified fatty acids and liver triglyceride percentages and decreased plasma IGF-I concentrations. The decrease in plasma IGF-I after feed restriction was associated with a decrease in IGF-I mRNA in feed-restricted cows. The amount of GHR 1A mRNA did not change after feed restriction. Epinephrine treatment did not affect the amount of GHR 1A or IGF-I mRNA. We conclude that the decrease in feed intake at parturition may be partially responsible for the decrease in IGF-I but may not cause the decrease in GHR 1A.     

Developmental and nutritional regulation of the prepubertal bovine mammary gland: II. Epithelial cell proliferation, parenchymal accretion rate, and allometric growth

It is well documented that elevated nutrient intake prior to puberty reduces prepubertal mammary development in the bovine. The companion paper demonstrated that age at harvest is a primary determinant of parenchymal (PAR) mass and that any effects of elevated energy intake on mechanisms regulating mammary development are dwarfed by this effect of time. Therefore, it is hypothesized that while causing a decrease in prepubertal PAR mass, elevated nutrient intake will have no effect on growth characteristics of the mammary gland. The objectives of this experiment were to evaluate the effects of increased nutrient intake from early in life on 1) mammary epithelial cell proliferation, 2) mammary PAR DNA accretion rates, and 3) the dynamics of prepubertal allometric PAR growth. Holstein heifers (n = 78) were fed from 45 kg of body weight either elevated (E) or restricted (R) levels of nutrients to support 950 (E) or 650 (R) g/d of body weight gain. Six heifers per treatment were harvested at 50-kg increments from 100 to 350 kg of body weight. Heifers on the E plane of nutrition had higher plasma leptin and less PAR DNA than their body weight-matched R-intake cohorts. Despite this reduction in PAR DNA, treatment did not negatively influence mammary epithelial cell proliferation or the PAR DNA accretion rate. Dynamics of allometric and isometric mammary growth were also unaffected by the level of nutrient intake, as was exit from allometric growth. This work represents the first demonstrating that the level of nutrient intake and the concomitant increase in plasma leptin have no measurable influence on 1) the rate of PAR DNA accretion, 2) mammary epithelial cell proliferation, or 3) total PAR mass and, by default, the local or systemic controls that coordinate these processes.