In prepubertal cattle, mammary development is characterized by the growth of an epithelial-rich parenchyma (PAR) into the mammary fat pad (MFP). This proliferation and accumulation of mammary epithelial cells require estrogen. Paradoxically, both epithelial cell proliferation and PAR accumulation rate decline with rising plasma estrogen as puberty approaches. The possibility that variation in abundance of estrogen receptors (ERs) in PAR or MFP accounts for a portion of these effects has not been examined in cattle. Additionally, we recently demonstrated that MFP is highly responsive to exogenous estrogen, suggesting that this tissue may play a role in coordinating estrogen’s effects on PAR; however, the developing bovine MFP has yet to be studied in detail. To address these hypotheses, Holstein heifers were assigned to planes of nutrition supporting body growth rates of 950 (E) or 650 (R) g/day and harvested every 50 kg from 100 to 350 kg body weight (BW). Post-harvest, their mammary glands were dissected into PAR and MFP compartments. Transcript abundance of genes encoding members of the ER family (ERα, ERβ, and estrogen-related receptor α-1 (ERRα)) and estrogen-responsive genes (IGF-I and progesterone receptor (PR)) were measured in both mammary compartments by quantitative real-time RT-PCR. Significant expression was detected for all genes in both compartments, with the exception of the ERβ gene. Transcript abundance of both ERα and IGF-I decreased linearly with increasing BW within both compartments. ERRα and PR expressions decreased with increasing BW in PAR but not in MFP. Nutrition stimulated ERα and ERRα expression in the PAR but had no effect on IGF-I or PR in either PAR or MFP. Overall, ERα and IGF-I transcript abundance are consistent with the drop in mammary epithelial cell proliferation and PAR accretion observed over development, but do not support a negative effect of nutrition on PAR growth.
M J Meyer, R P Rhoads, A V Capuco, E E Connor, A Hummel, Y R Boisclair and M E Van Amburgh
H El Sheikh Saad, A Toullec, S Vacher, M Pocard, I Bieche and M Perrot-Applanat
Exposure to low doses of environmental estrogens such as bisphenol A and genistein (G) alters mammary gland development. The effects of environmental anti-androgens, such as the fungicide vinclozolin (V), on mammary gland morphogenesis are unknown. We previously reported that perinatal exposure to G, V, and the GV combination causes histological changes in the mammary gland during the peripubertal period, suggesting alterations to the peripubertal hormone response. We now investigate whether perinatal exposure to these compounds alters the gene expression profiles of the developing glands to identify the dysregulated signaling pathways and the underlying mechanisms. G, V, or GV (1 mg/kg body weight per day) was added to diet of Wistar rats, from conception to weaning; female offspring mammary glands were collected at postnatal days (PNDs) 35 and 50. Genes displaying differential expression and belonging to different functional categories were validated by quantitative PCR and immunocytochemistry. At PND35, G had little effect; the slight changes noted were in genes related to morphogenesis. The changes following exposure to V concerned the functional categories associated with development (Cldn1, Krt17, and Sprr1a), carbohydrate metabolism, and steroidogenesis. The GV mixture upregulated genes (Krt17, Pvalb, and Tnni2) involved in muscle development, indicating effects on myoepithelial cells during mammary gland morphogenesis. Importantly, at PND50, cycling females exposed to GV showed an increase in the expression of genes (Csn2, Wap, and Elf5) related to differentiation, consistent with the previously reported abnormal lobuloalveolar development previously described. Thus, perinatal exposure to GV alters the mammary gland hormone response differently at PND35 (puberty) and in animals with established cycles.
SD Berry, RD Howard, PM Jobst, H Jiang and RM Akers
The objective was to determine the effects of ovariectomy and epithelial-stromal interactions on mammary development and local expression of IGF-I and IGF-binding protein (IGFBP) mRNA in prepubertal heifers. An epithelium-free ('cleared') fat pad (CFP) was prepared in two glands in each of 14 Holstein heifers, aged 1-3 Months. Eight of the calves were also ovariectomized. Serum concentrations of GH, IGF-I and prolactin were not affected by ovariectomy. At 6 Months of age, calves were killed to provide mammary samples of parenchyma, CFP and intact fat pad (MFP). Total mammary mass was reduced in ovariectomized calves (130+/-21 g vs 304+/- 25 g; P<0.001), and in several cases parenchymal tIssue was essentially absent. Uterus weight was also reduced by ovariectomy (14.5+/-3.8 g vs 30.4+/-4.5 g; P<0.05). In support of our hypothesis that local IGF-I mediates prepubertal mammary development, mRNA expression of IGF-I was lower in ovariectomized than in control calves (62.1+/-7.8 vs 91.6+/-7.8 arbitrary units; P<0.05). Specific binding of IGF-I to mammary parenchymal microsomes was also reduced by ovariectomy (377+/-142 vs 868+/-82 c.p.m.; P<0.01), suggesting decreased sensitivity to IGF-I. Expression of IGFBP-3 and IGFBP-5 mRNA were not influenced by ovariectomy. Expression of IGF-I, IGFBP-3 and IGFBP-5 mRNA did not differ between CFP and MFP, suggesting that expression of these factors was not influenced by interactions between stroma and developing epithelium. Overall, the data suggested that interactions between the ovary and the local IGF-I axis act to optimize the availability and effectiveness of IGF-I within the gland to stimulate mammary growth.
V Sriraman, MR Sairam and AJ Rao
The relative role of LH and FSH in regulation of differentiation of Leydig cells was assessed using an ethane 1,2-dimethylsulfonate (EDS)-treated rat model in which endogenous LH or FSH was neutralized from day 3 to day 22 following EDS treatment. Serum testosterone and the in vitro response of the purified Leydig cells to human chorionic gonadotropin (hCG) was monitored. In addition RNA was isolated from the Leydig cells to monitor the steady-state mRNA levels by RT-PCR for 17alpha-hydroxylase, side chain cleavage enzyme, steroidogenic acute regulatory protein (StAR), LH receptor, estrogen receptor (ER-alpha) and cyclophilin (internal control). Serum testosterone was undetected and the isolated Leydig cells secreted negligible amount of testosterone on stimulation with hCG in the group of rats that were treated with LH antiserum following EDS treatment. RT-PCR analysis revealed the absence of message for cholesterol side chain cleavage enzyme and 17alpha-hydroxylase although ER-alpha and LH receptor mRNA could be detected, indicating the presence of undifferentiated precursor Leydig cells. In contrast, the effects following deprival of endogenous FSH were not as drastic as seen following LH neutralization. Deprival of endogenous FSH in EDS-treated rats led to a significant decrease in serum testosterone and in vitro response to hCG by the Leydig cells. Also, there was a significant decrease in the steady-state mRNA levels of 17alpha-hydroxylase, cholesterol side chain cleavage enzyme, LH receptor and StAR as assessed by a semiquantitative RT-PCR. These results establish that while LH is obligatory for the functional differentiation of Leydig cells, repopulation of precursor Leydig cells is independent of LH, and also unequivocally establish an important role for FSH in regulation of Leydig cell function.
J Tsubaki, WK Choi, AR Ingermann, SM Twigg, HS Kim, RG Rosenfeld and Y Oh
Dietary factors play an important role in both the development and prevention of human cancers, including breast carcinoma. One dietary micronutrient, sodium butyrate (NaB), is a major end product of dietary starch and fiber, produced naturally during digestion by anaerobic bacteria in the cecum and colon. NaB is a potent growth inhibitor and initiates cell differentiation for many cell types in vitro. In this study, we investigated the effects of NaB on three human mammary epithelial cells and regulation of the IGF axis, specifically, IGF-binding protein-3 (IGFBP-3), a known growth regulator in human mammary cells, and IGFBP-related protein 2 (IGFBP-rP2)/connective tissue growth factor. NaB inhibited DNA synthesis, as measured by [3H]thymidine incorporation, in estrogen-responsive (MCF-7) and estrogen-non-responsive (Hs578T) breast cancer cells, and normal human mammary epithelial cells (HMEC) to a similar degree (up to 90% inhibition at 1-10 mM concentrations). Treatment of cells with NaB induced histone hyperacetylation, suggesting that NaB exerts its biological effects, at least in part, as a histone deacetylase inhibitor in mammary epithelial cells. Treatment of Hs578T cells with NaB caused an induction of apoptotic cell death. NaB treatment resulted in increased levels of p21(Waf1/Cip1) mRNA and protein in Hs578T cells and distinct upregulation of p27(Kip1) in HMEC, suggesting that NaB activates different genes involved in cell cycle arrest, depending upon the cell type. In the same context, among the IGFBP superfamily members tested, NaB specifically upregulated the expression of IGFBP-3 and IGFBP-rP2. These two proteins are known to be involved in inhibition of mammary epithelial cell replication. Northern blot analysis showed that NaB treatment at 1-10 mM concentrations caused a dose-dependent stimulation of IGFBP-3 mRNA expression in cancerous cells and IGFBP-rP2 mRNA expression in both cancerous and non-cancerous cells. Protein data from Western ligand blot and immunoblot analyses demonstrated parallel results. In summary, we have demonstrated that NaB (i) uniformly suppresses DNA synthesis in both cancerous and non-cancerous mammary cells, and (ii) upregulates IGFBP-3 and IGFBP-rP2 mRNA and protein levels in cancerous and non-cancerous mammary cells. These results provide the first demonstration that butyrate regulates the IGFBP system in the human mammary system.
M. A. SAJI and D. B. CRIGHTON
A rabbit antiserum to a purified preparation of ovine prolactin was prepared. The capacity of the antiserum to counter the biological effect of the preparation of ovine prolactin was determined. When injected intraductally before the injection of prolactin into the same duct the antiserum inhibited the lactogenic effect of prolactin on the rabbit mammary gland. The weight, nitrogen content and reducing sugar content of the mammary glands were used as criteria to judge the effect of the antiserum. The results of specificity tests on the antiserum, using the techniques of double diffusion and immunoelectrophoresis, are also reported.
REIKO YANAI and HIROSHI NAGASAWA
The effects of 2-hydroxyoestradiol-17β and 2-hydroxyoestriol (2OH-OE3) on levels of serum prolactin, DNA synthesis by the mammary gland and the weight of the uterus were examined in ovariectomized Sprague–Dawley rats.
2-Hydroxyoestradiol-17β had a potent oestrogenic activity in stimulating the secretion of prolactin by the pituitary gland, the synthesis of DNA by the mammary gland and in increasing the weight of the uterus. On the other hand, 2OH-OE3 increased the weight of the uterus only.
P Guillaumot and H Cohen
This work was undertaken to determine variations in the 125I-labelled ovine prolactin (oPRL) binding in rat liver and mammary gland membranes, and to study the molecular forms of prolactin receptor in different physiological situations. Prolactin binding was determined using 125I-labelled oPRL in the 100 000 g pellet. 125I-Labelled oPRL was cross-linked to receptors in membranes from rat liver and mammary gland and subjected to SDS-PAGE under reducing conditions, followed by autoradiography of dried gels.
In the mammary gland, the specific binding of oPRL to membranes, expressed as mean ± s.e.m. fmol/mg protein increased from 1·36 ±0·24 on the day of dioestrus to 3·26 ±0·60 on the day of oestrus. It remained very low during pregnancy but increased during lactation to reach 4·78 ±0·99. In the liver, the specific binding of oPRL to membranes was higher than in the mammary gland on the days of dioestrus 1, dioestrus 2 and oestrus, but not on the day of pro-oestrus. It increased until day 14 of pregnancy when the specific binding of 125I-labelled oPRL was 17·01 ±0·30.
Cross-linking revealed different molecular forms in the mammary glands and the liver. In the mammary gland we observed four prolactin-binding forms of 80, 50, 40 and 16 kDa, all of which were specific for prolactin. The 80, 50 and 40 kDa forms were also able to bind to a Concanavalin A–Sepharose column, indicating that these binding forms were glycosylated while the smaller one (16 kDa) appeared to be unglycosylated. The 40 kDa prolactin receptor was seen at all stages studied: the oestrous cycle (dioestrus, pro-oestrus and oestrus), pregnancy (days 8, 14 and 22) and lactation. The 50 kDa form was observed in the mammary gland during the day of pro-oestrus and gestation. It was also observed in ovariectomized rats treated with oestradiol (OE2), suggesting that OE2 could be one of the factors involved in the induction of this receptor form. The 16 kDa form of the receptor was found in the mammary gland only during lactation. This form, while unglycosylated, bound specifically to prolactin, suggesting that it might play a specific role during lactation.
In the liver, two forms were shown, a major one of 40 kDa and a minor one of 80 kDa. No variation in receptor molecular weight was found in the liver during the physiological stages studied.
Journal of Endocrinology (1994) 141, 271–278
A. T. COWIE, G. S. KNAGGS, J. S. TINDAL and A. TURVEY
The regular twice daily 'milking' of three ovariectomized virgin goats brought about mammary growth and the initiation of lactation. After several months daily milk yields of 0·8 to 2·0 1. were attained. The mammogenic and lactogenic responses to the milking stimulus were completely abolished in four virgin goats (two ovariectomized and two with intact ovaries) in which the pituitary stalk had been transected before 'milking' was started. The neurohormonal mechanisms concerned and the significance of these observations for the interpretation of studies on the hormonal induction of mammary growth and lactation are discussed.
T. R. BRADLEY and A. T. COWIE
Mammary gland slices (incubated in vitro) from hypophysectomized rats used less glucose and produced more lactic acid than similar slices from normal animals. The accumulation of lactic acid was observed in the presence of glucose or glucose plus acetate as added substrates, but not in acetate alone or in the absence of added substrates. Hypophysectomy led to a substantial decrease in the respiratory quotient of the mammary gland slices. Neither ovariectomy, adrenalectomy, thyroidectomy, parathyroidectomy nor sham hypophysectomy caused the appearance of the above metabolic changes in mammary gland slices.