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SUMMARY
Explants of mammary glands and of subcutaneous body fat from sexually mature virgin and from 19-day pregnant Sprague-Dawley rats and of mammary gland from 5-day lactating Sprague-Dawley rats, were maintained in organ culture for up to 96 h. The effects of insulin (I), corticosterone (B), prolactin (P) and growth hormone (G) on the rate of fatty acid synthesis were measured by the incorporation of [14C]acetate. The effect of these hormones on the synthesis of various carbon-chain length fatty acids was measured by radio gas-liquid chromatography.
Explants from both tissues had a reduced rate of fatty acid synthesis after 24 h in medium 199, but this rate was increased by the addition of insulin. In explants of subcutaneous fat from virgin rats, the rate was further increased by culture in IBP or IBG, but this increase was not blocked by actinomycin D. In explants from subcutaneous fat of 10-day pregnant rats the rate was not increased by the addition of B, P or G to the insulin-containing medium. In mammary gland explants from virgin rats, IBP stimulated a greater rate of fatty acid synthesis than did insulin alone. In mammary gland explants from 10-day pregnant rats, the rate of fatty acid synthesis was increased by both IBP and IBG. In mammary gland explants from rats on the 5th day of lactation, both IBP and IBG increased the rate of fatty acid synthesis compared with insulin or IB. Actinomycin D blocked the increased fatty acid synthesis produced by prolactin or growth hormone but not that produced by insulin alone.
Mammary gland explants from rats on the 5th day of lactation were cultured for the first 4 h after excision in medium 199 that contained sodium [14C]acetate. Sixty-eight per cent of the 14C was incorporated into C8-C12 fatty acids. In explants from subcutaneous fat none of the hormones tested increased 14C incorporation in these fatty acids. In mammary gland explants from virgin or 10-day pregnant rats, insulin, corticosterone and prolactin increased the incorporation in these fatty acids. Growth hormone was less efficient than prolactin in stimulating C8-C12 fatty acid synthesis.
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SUMMARY
The effect of various hormones on the incorporation of [14C]acetate into the fatty acids of pregnant mouse mammary gland explants in organ culture was studied.
Of the hormones insulin (I), ovine prolactin (P), bovine growth hormone (GH) and cortisol (F) tested singly, only insulin stimulated fatty acid synthesis. There was synergism between cortisol or prolactin with insulin. The greatest stimulation in fatty acid synthesis occurred when explants were incubated in a medium containing either I + F + P or I + F + GH.
Analysis by radio-gas-liquid chromatography of the fatty acids synthesized by explants after 14C labelling, showed that the pattern of fatty acids formed in the presence of I + F was distinctly different from that produced in the presence of I + F + P or I + F + GH. In the presence of I + F, the pattern of fatty acids resembled that found in mouse adipose tissue, whilst with I + F + P or I + F + GH the pattern resembled that of mouse milk fat.
Synthesis of RNA was essential for the stimulation of fatty acid synthesis in explants incubated in medium containing I + F + P or I + F + GH. Results obtained when DNA synthesis was blocked with mitomycin C suggest that mitosis is important for the induction of milk-fatty acid synthesis.
Puromycin had no effect for up to 8 h on explants which had been previously cultured in medium containing I + F, I + F + P or I + F + GH. This suggests a slow turnover rate of the enzymes involved in the synthesis of milk fatty acids.
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Multi-alveolar mammary structures (mammary lobules) were prepared from mammary glands of pseudopregnant rabbits by controlled digestion with collagenase and hyaluronidase. The overall rate of fatty acid synthesis and the proportion of milk-specific fatty acids (C8:0 and C10:0) synthesized by these lobules when cultured with insulin, corticosterone and prolactin were measured. Maximum response to physiological concentrations of prolactin (1·1 or 2·2 nmol/l) occurred in the presence of insulin (1·7 μmol/l) and corticosterone (0·58 μmol/l). In general, the results obtained on the effect of progesterone were negative. Though explants showed a ninefold greater response to prolactin per mg DNA than did mammary lobules, the latter have the advantage of being easily prepared for culture in large numbers. Reduction to below 500 μm diameter and culture in conditions which allow cell outgrowth onto plastic limited their response to prolactin. The probable roles of membrane damage by digesting enzymes and of tissue architecture in limiting prolactin response are discussed.