Lactogenesis in Explant Cultures of Mammary Tissue from Pregnant Cows1 R. J. COLLIER,2 D. E. BAUMAN, AND R. L. HAYS Department of Dairy Science, University of Illinois, Urbana, Illinois 61801 ABSTRACT. The hormonal requirements for lactogenesis were investigated using explant cultures of mammary tissue obtained from cows at 30-40 days prepartum. Hormones used were insulin, hydrocortisone, and prolactin, and parameters examined were radioactive acetate incorporation into fatty acids, secretory response ratings, and histological and ultrastructural analysis. Data indicated that insulin was essential for mammary epithelial cell survival, but insulin alone did not result in the initiation of milk synthesis. The culture of explants in a medium containing insulin plus hydrocortisone resulted in alterations in the cytology of alveolar cells but no induction of milk synthesis. Biosynthesis results and secretory response ratings indicated that the initiation of milk synthesis occurred when explants were cultured in insulin and prolactin; however, synthesis was limited and alveolar integrity

was not well maintained. Results from all parameters demonstrated that the maximal lactogenic response was obtained when the culture medium contained insulin, hydrocortisone and prolactin. After 48 h of culture in this medium, the rate of acetate incorporation into fatty acids had increased 3-fold and the markedly distended alveolar lumina contained many fat droplets and abundant eosinophilic staining secretion. However, an unusual amount of casein-like micelles and especially lipid also accumulated in the alveolar cells. The accumulation of milk components within the epithelial cells may be related indirectly to the accumulation of products in the lumina or perhaps related to a difference in the hormonal requirement between the initiation of milk synthesis and initiation of milk secretion. (Endocrinology 100: 1192, 1977)

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ACTOGENESIS, the initiation of milk species and demonstrated that lactogenesis synthesis, results in dramatic bio- occurred when mammary explants were culchemical and cytological changes in mam- tured in a milieu containing insulin, a mary epithelial cells. Although relationships corticoid, and prolactin (2,4-6). between endocrine factors and the mamRelatively few investigations have exmary tissue events in the initiation of milk amined mammary tissue from ruminants. synthesis are not well understood, several Using mammary explants obtained from excellent reviews have summarized the cur- pregnant (day 30) sheep, Jeulin-Bailly et al. rent knowledge (1-3). In vivo studies in- (7) have examined the hormonal requirevolving removal of various endocrine glands ments for growth and the development and/or exogeneous hormone administration of secretory activity (histological ratings). have indicated that the hormonal control of In view of species differences noted with lactogenesis appears to differ among in vivo studies, our objective was to examine species. In recent years, mammary explant the hormonal requirements for lactogenesis cultures have been utilized to allow a more in explant cultures of mammary tissue obprecise identification of specific hormone tained from cows in late pregnancy. While actions and/or multiple hormone interac- our investigations were in progress, short tions in the initiation of milk synthesis. communications were published with a These in vitro investigations have utilized similar objective in which mammary tissue mammary tissue from small laboratory explants from nonpregnant heifers (8) and pregnant (9-10 weeks) goats (9) were utilized. Received October 12, 1976. 1 Supported in part by funds from the Illinois Agricultural Experiment Station. 2 Present address: Department of Dairy Science, University of Florida, Gainesville, Florida 32611. Address Correspondence to: D. E. Bauman.

Materials and Methods Mammary tissue was obtained from 4 pregnant, multiparous Holstein cows by surgical

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LACTOGENESIS IN COW MAMMARY EXPLANTS biopsy as previously described (10). Cows were 30-40 days prepartum and had not been lactating for 22-44 days at time of biopsy. The mammary samples (10-15 g) were immediately placed in sterile isotonic sucrose until explants were prepared (approximately 5-10 min). Methods for the preparation of mammary explants and the culture incubations were essentially as described by Topper et al. (11). Explants were incubated in plastic culture dishes (#3010, Falcon Plastics Co., Oxnard, Ca.) and the basal medium was Tissue Culture Medium 199 with Hanks Salts (Difco Laboratories, Detroit, Mich.). Antibiotics included in the basal medium were 10 /xg/ml streptomycin sulfate (Eli Lilly Corp., Indianapolis, In.), 100 units/ml potassium penicillin-G (E. R. Squibb and Sons, Inc., New York, N.Y.). In addition, the concentration of acetate in the basal medium was raised to 10 mM since previous investigations have shown that this concentration is required to sustain metabolic rates in incubations of cow mammary tissue slices (12). Hormones added to the culture medium in various combinations were insulin (I), hydrocortisone (C) and prolactin (PRL). Insulin (Sigma Chemical. Co., St. Louis, Mo.) was first dissolved in sterile 0.005N HC1 then added to the medium to reach a final concentration of 5 jug/ml. Bovine prolactin (NIH-P-B3; National Institute of Health, Bethesda, Md.) was dissolved in sterile 0 . 0 0 1 N NaOH solution and added to the culture media to a final concentration of 1 /xg/ml. Hydrocortisone (Sigma Chemical Co.) was dissolved in absolute ethanol then added to culture media to reach a concentration of 5 /tig/ml. The final concentration of ethanol in the culture media did not exceed 0.5%. Ethanol concentrations below 0.5% in media have no deleterious effects on mammary tissue in explant cultures (11). In the preparation of the explants and the culture incubations, care was taken to maintain aseptic conditions. The basal culture medium was sterilized by Millipore filtration and then the various hormones were added. For the explant incubations, culture dishes were placed in desiccators, gassed with a mixture of 95% O2 and 5% CO2, and placed in an oven at 37 C. At zero time and following various intervals of culture, explants were removed and either fixed for histological or ultrastructural analysis or placed in a biosynthesis medium to determine rates of fatty acid synthesis.

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Methods of fixation, sectioning, staining and microscopy of the tissue were described by Croom et al. (13). The histological sections were also prepared for secretory response ratings using the grading system described by Barnawell (4). The incubation medium (1 ml volume) for biosynthetic studies was composed of Krebs-Ringer bicarbonate buffer (pH 7.3), 10 mM acetate, 10 mM glucose, 133 munits/ml of insulin and 0.3 /x,Ci/ml of [2-14C]acetate as previously described (14). Following 2 h of incubation at 37 C in a shaking water bath in an atmosphere of 95% O2 and 5% CO2, the reactions were terminated with the addition of 0.1 ml of IN H2SO4 to the media. Lipids present in tissue explants were saponified, extracted, and quantitated as described by Bauman et al. (14). Statistical analysis was by one-way analysis of variance. Results Biosynthetic

studies

Acetate is the major substrate utilized by cow mammary tissue for the de novo synthesis of milk fatty acids (14). The rates of acetate incorporation into fatty acids by mammary explants following culture with various hormone combinations are shown in Fig. 1. Explants were not cultured in medium containing only hydrocortisone or prolactin because previous investigations with mice have demonstrated a poor survival of mammary explants for these media (6). In our investigations, explants were cultured for various time periods (24, 48 or 72 h), then removed from the culture and incubated in the biosynthetic medium for 2 h to measure the capacity of the explant to incorporate radioactive acetate into fatty acids. At time zero, when the tissue was removed from the animal, the rate of acetate incorporation into fatty acids was 1.34 nmol/mg tissue per 2 h (Fig. 1). This rate represents 40-80% of the rates obtained by Mellenberger et al. (10) (tissue slice incubations) and Kinsella (15) (tissue homogenate incubations) for cow mammary tissue obtained during late pregnancy. These investigators further demonstrated that this relatively low

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Endo • 1977 Vol 100 • No 4

COLLIER, BAUMAN AND HAYS

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ICPrl

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FIG. 1. Acetate incorporation into fatty acids by mammary explants from pregnant cows following culture with various hormone combinations. Explants were cultured with no hormones (O) or various combinations of insulin (I), hydrocortisone (C) and prolactin (Prl). At time periods indicated, explants were incubated for 2 h in a biosynthesis medium containing radioactive acetate. Each value represents mean of 9 determinations from 3 cows.

acetate incorporation into fatty acids represented synthesis of cellular lipids rather than milk lipids (10,15). The culture of mammary explants in the absence of any hormones resulted in a steady decline in the lipogenic rate and by 72 h of culture the rate of fatty acid synthesis was approximately 60% of the zero time value (Fig. 1). When cultured in insulin medium or insulin plus hydrocortisone medium, there was a slight increase in the rate of synthesis followed by a decrease. In contrast, when mammary explants were cultured in the presence of insulin plus prolactin medium or insulin, hydrocortisone and prolactin medium there was a dramatic increase in the rate of fatty acid synthesis. The combination of hormones having the greatest effect on fatty acid synthesis rates was insulin, hydrocortisone and prolactin. At 48 h of culture in ICPRL, the lipogenic rate was 3 times greater than the initial zero time value. Several methods for determining the rates of glucose incorporation into lactose were

also examined. Due to the small quantity of tissue in each culture (5 to 10 mg) it was extremely difficult to quantitate lactose biosynthesis. Limited success was attained using an enzymatic method (10). The minimum concentration of lactose which this spectrophotometric assay could detect was 3 fig/ml of culture medium. Culture media from experiments shown in Fig. 1 were examined. Only media from explants cultured for 48 or 72 h in ICPRL had detectable lactose present. At 72 h of culture, the lactose concentration in this culture medium averaged 5.8 fig/ml. In a study involving mammary tissue obtained from one pregnant cow, explants were cultured for 48 h in media containing various hormone combinations and then switched to ICPRL medium for an additional 72 h (Fig. 2). For the first 48 h of culture the results in the various media were similar to the previous study. After transfer to ICPRL, explants cultured initially in IC showed a dramatic increase in the rates of fatty acid 5rICPrl y

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Lactogenesis in explant cultures of mammary tissue from pregnant cows.

Lactogenesis in Explant Cultures of Mammary Tissue from Pregnant Cows1 R. J. COLLIER,2 D. E. BAUMAN, AND R. L. HAYS Department of Dairy Science, Unive...
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