Section A. Physiology. Organ culture techniques and the study of hormone effects on the mammary gland

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 mumol/l) and corticosterone (0.58 mumol/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 microns 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.

Section: Discussionsupporting

confidence: 83%

Milk-fat synthesis by lobules prepared from rabbit mammary gland: response to insulin, corticosterone, prolactin and progesterone

Carrington¹,

Hosick²,

Forsyth³

et al. 1983

“…Cortisol is synergistic with prolactin (Denamur, 1971 ;Forsyth, 1971) and its concentration increases in late pregnancy (Mulay et al 1973) in parallel with the level of prolactin at the time when milk synthesis increases (days 26-31). The concentration of progesterone which is known to depress lactogenesis (Kuhn, 1969;Turkington & Hill, 1969;Assairi, Delouis, Gaye, Houdebine, Ollivier-Bousquet & Denamur, 1974) appears to decrease rather early in the rabbit (Challis et al 1973), the dog (Hardley, 1975), the cat (Verhage, Beamer & Brenner, 1976) and the pig (Robertson & King, 1974), all species in which the occurrence of placental lactogenic hormones remains doubtful (Kelly et al 1976).…”

Section: Discussionmentioning

confidence: 99%

Lactogenic Activity in the Serum of Rabbits During Pregnancy and Early Lactation

Durand¹,

Djiane²

1977

The levels of lactogenic activity in the serum of rabbits during pregnancy and early lactation have been determined. The accuracy and sensitivity of the radioreceptor assay used were increased by several technical improvements. Lactogenic activity remained low throughout pregnancy and increased at parturition; the highest values were reached on day 5 of lactation. No lactogenic activity was detected in placental extracts of rabbits, suggesting that the lactogenic activity measured in the serum is solely of pituitary origin. These results are discussed in relation to the main stages of development of rabbit mammary glands.

“…The enlarged lumina were filled with an eosinophilic secretion containing numerous fat droplets. The addition of ovine growth hormone (NIH-GH-S10, 0-86 i.u./mg, 5 Mg/ml) did not modify the response observed with insulin + cortisol + prolactin, while insulin + prolactin did not induce milk secretion in vitro.As in most mammals (see Denamur, 1969Denamur, , 1971Forsyth, 1971) insulin + cortisol + prolactin is thus the minimal hormonal requirement for lactogenesis in bovine mammary tissue in vitro. Histological results correlated well with those obtained on the appearance of lactose synthetase activity during lactogenesis in heifer mammary tissue in organ culture (J. Djiane &C. Delouis, unpublished observations).…”

mentioning

confidence: 92%

“…As in most mammals (see Denamur, 1969Denamur, , 1971Forsyth, 1971) insulin + cortisol + prolactin is thus the minimal hormonal requirement for lactogenesis in bovine mammary tissue in vitro. Histological results correlated well with those obtained on the appearance of lactose synthetase activity during lactogenesis in heifer mammary tissue in organ culture (J. Djiane &C. Delouis, unpublished observations).…”

mentioning

confidence: 92%

Lactogenesis in Organ Cultures of Heifer Mammary Tissue

Djiane¹,

Delouis²,

Denamur³

1975

Hormonal induction of milk synthesis by the ruminant mammary gland in organ culture has received little attention. M. A. Cannata, C. Delouis, C. Jeulin & R. Denamur (unpublished observation) demonstrated the lactogenic effect of insulin + cortisol + prolactin in vitro on ewe mammary tissue with lobulo-alveolar development at the time of explantation, but there are no data on the induction of milk secretion in bovine mammary tissue cultured on a synthetic medium. Therefore, we have studied the culture conditions necessary to maintain bovine mammary tissue and the combination of hormones needed to induce milk secretion.Biopsy samples were obtained from 2-year-old French Friesian heifers at various times during the oestrous cycle, after i.m. injection of the tranquillizer, xylazine (Rompun, Bayer; 2% solution, 1*5 ml/100 kg body weight). Expiants (1 mm3) were cultured in organ culture dishes (Falcon Plastics). The culture medium was medium 199 (Microbiological Associates) and Eagle's minimum essential medium (MEM, Eurobio) in equal amounts. The tissue was not so well maintained on Medium 199 or MEM alone. Final amino acid concentration was adjusted to five times that of Medium 199 by adding a concentrated amino acid solution (Eagle's basal medium amino acids, 100, Microbiological Associates); sodium bicarbonate (2-2 g/1) and sodium acetate (5-44 g/1) were also added to the medium. Cultures were incubated at 37°C in 57 % 02:5 % C02:38 % N2 (by vol.).Histological examination of biopsy samples revealed poorly developed mammary tissue; in most cases ducts with small end buds only were present. Cultured on medium containing amorphous insulin (Endopancrine, 24-5 i.u./mg; 5 ug/ml) this tissue was well maintained as judged by light microscopy for up to 10 days. In an atmosphere of 95 % 02:5 % C02 (v/v) maintenance was very poor, even if the medium contained cortisol (Microfine, Roussel; 5^g /ml) in addition to insulin and/or 20 % serum from hypophysectomized ewes. These results agreed well with observations of M. A. Cannata et al. (unpublished results) on the survival of pregnant ewe mammary tissue in vitro. Bovine mammary tissue was also well maintained on a synthetic medium supplemented with insulin if the atmosphere was 95 % air : 5 % C02, but the cells of ducts cut across during the preparation of the tissue for culture tended to migrate around the surface of the expiants.Results presented in Table 1 show that insulin + cortisol did not induce secretion after 5 days in vitro. Addition of ovine prolactin (NIH-P-S9, 30 i.u./mg, 5 Mg/ml) to this medium induced secretion in most of the expiants. Epithelial cells had abundant cytoplasm and their nuclei were basal. The enlarged lumina were filled with an eosinophilic secretion containing numerous fat droplets. The addition of ovine growth hormone (NIH-GH-S10, 0-86 i.u./mg, 5 Mg/ml) did not modify the response observed with insulin + cortisol + prolactin, while insulin + prolactin did not induce milk secretion in vitro.As in most mammals (see Denamur, 1969Denamur, , 1971Forsyt...