Rabbit whey acidic protein has been purified from whey using an AcA54 column. The purified whey acidic protein had an amino acid composition in agreement with the previously defined cDNA sequence. An antibody against whey acidic protein was raised in guinea pig. This antibody did not crossreact with mouse or cow milk or with rabbit alpha s1-casein and beta-casein. Whey acidic protein concentration was measured in rabbit milk using the antibody with a radioimmunoassay. The concentration of whey acidic protein in rabbit milk was 15 mg/ml, whereas the concentrations of alpha s1-casein and beta-casein were 16 and 45 mg/ml, respectively. The concentration of the three proteins was also evaluated in culture medium of rabbit primary mammary cells. The three proteins were induced by prolactin alone. Glucocorticoids amplified the prolactin effect on whey acidic protein more intensively than on caseins. The three proteins were present in mammary extract from virgin rabbit. The concentration of these proteins was lower at d 8 and 14 of pregnancy, and it was very high at d 25 of pregnancy. Whey acidic protein was undetectable in blood of virgin, weaned, and midpregnant females and of males. Whey acidic protein was present in blood of lactating rabbits, but alpha s1-casein and beta-casein were not detectably present in rabbit blood at the examined physiological states.

No abstract

No abstract

The 5' flanking region (6.3 kb) of the rabbit WAP (rWAP) gene possesses important regulatory elements. This region was linked to the human growth hormone (hGH) structural gene in order to target transgene expression to the mammary gland. Thirteen lines of transgenic mice were produced. Milk could be collected from six lines of transgenic mice. In five of them, hGH was present in the milk at high concentrations ranging from 4 to 22 mg ml-1. hGH produced by the mammary gland comigrated with hGH of human origin. It was biologically active, and through its prolactin-like activity induced lactogenesis when introduced into mammary culture media. Two of these mouse lines were studied further. hGH mRNA was only detected in the mammary gland during lactation. In the seven other transgenic lines, hGH was present in the blood of cyclic females. The prolactin-like effect of hGH in these mice probably induced female sterility, and milk could therefore not be obtained. In two lines studied in more detail, the mammary gland was the main organ producing hGH, even in cyclic mice. Low ectopic expression was detected in other organs which varied from one line to the other. This was probably due to the influence on the transgene of the site of integration into the mouse genome. In the 13 lines studied, high mammary-specific hGH expression was not correlated to the transgene copy number. The rWAP-hGH construct thus did not behave as an independent unit of transcription. However, it can be concluded that the 6.3 kb flanking region of the rWAP gene contains regulatory elements responsible for the strong mammary-specific expression of hGH transgene, and that it is a good candidate to control high levels of foreign protein gene expression in the mammary gland of lactating transgenic animals.

The concentration of transferrin mRNA was evaluated during pregnancy and lactation in rabbit mammary gland and liver using northern blot and dot blot assays. Transferrin mRNA was present in the virgin rabbit mammary gland and its concentration increased as pregnancy proceeded, with a major enhancement after day 15. A high concentration was reached 3 days after parturition, with no additional increase during lactation and with a marked decline after weaning. During the same period, the concentration of transferrin mRNA showed only a very weak variation in liver. This mRNA was six times more abundant in mammary gland than in liver of lactating rabbit. The accumulation of transferrin mRNA in the mammary gland was concomitant with the accumulation of alpha s1-, beta-, kappa-casein and WAP (whey acidic protein) mRNAs. The concentration of glyceraldehyde 3-phosphate dehydrogenase mRNA, taken as a non-inducible control mRNA, declined progressively during pregnancy to reach its lower level in lactation. These observations suggest that casein, WAP and transferrin mRNAs are subjected to a similar control mechanism in vivo, at least in the second half of pregnancy and during lactation. Experiments carried out in vitro using isolated rabbit epithelial mammary cells cultured on collagen I gel indicated that transferrin mRNA was abundant and only weakly inducible by the lactogenic hormones insulin, cortisol and prolactin, as opposed to caseins and WAP mRNAs. R5020, an analogue of progesterone, inhibited at most very slightly the accumulation of alpha s1-casein mRNA in the presence of prolactin and it did not reduce the expression of transferrin gene.(ABSTRACT TRUNCATED AT 250 WORDS)