Progressive Changes in Plasma Progesterone, Prolactin and Corticosteroid Levels During Late Pregnancy and the Initiation of Lactose Synthesis in the Rat
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Aust. J. BioI. Sci., 1981, 34, 445-54 Progressive Changes in Plasma Progesterone, Prolactin and Corticosteroid Levels during Late Pregnancy and the Initiation of Lactose Synthesis in the Rat K. R. Nicholas A •B and P. E. HartmannA •C A Department of Biochemistry, University of Western Australia, Nedlands, W.A. 6009. B Present address: Laboratory of Biochemistry and Metabolism, National Institutes of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20205, U.S.A. C Address for reprints. Abstract The relationship between progesterone, prolactin, corticosteroids and corticosteroid binding globulin . (CBO) activity in plasma and the initiation of lactation were studied in normal parturient rats, and rats either ovariohysterectomized or Caesarean-sectioned on day 19 of gestation. In chronically cannulated rats the decline in plasma progesterone to low values « 10 p.g/l) in normal parturient rats 20 h before term and in Caesarean-sectioned rats 10--14 h after surgery was closely related to an increase in plasma prolactin. However, in ovariohysterectomized rats the levels of progesterone declined abruptly to 20 p.g/l within 30 min of surgery and prolactin remained low (5 p.g/l) for 4-8 h and then gradually increased during the subsequent 16-18 h. Lactose concentrations in mam mary tissue of rats killed at 0, 12, 18. 24, 36 and 48 h after surgery was low « 0·14 mg/g tissue) up to 12 h, and increased to reach maximum values at 36 hand 48 h after surgery in ovariohyster ectomized and Caesarean-sectioned rats respectively. The concentration of corticosteroids and CBO capacity in the plasma was 250--550 p.g/l and 250-480 p.g corticosterone bound per litre, respectively, in rats killed during the last 4 days of,gesta tion. However, both the concentration of corticosteroids and the CBO capacity, within the individual cannulated rats, remained relatively constant during late gestation. These findings support the proposal that progesterone withdrawal is the lactogenic trigger ana suggest the sequential involvement of prolactin. An increase in the concentration of free corticoster oids in late pregnancy and a related stimulatory role in the lactogenic mechanism were not established. Introduction Cowie (1969) outlined four general concepts concerning the control of initiation of lactation. These concepts were the inhibition of the effects of the lactogenic complex by progesterone and oestrogen, the completion of the lactogenic complex by an increase in the levels of the biologically active corticosteroids, oxytocin stimulation of prolactin release, and the neural stimulation of prolactin release. Subsequently, Kuhn (1969) reported that, in the rat, withdrawal of progesterone from the plasma preceded a rapid increase in the lactose content of the mammary tissue and that the initiation of lactose synthesis could be inhibited by the administration of progesterone. Therefore he concluded that progesterone withdrawal was the lactogenic trigger in the rat. However, the role of prolactin and glucocorticoids in the initiation oflactation in the rat remains unclear. Simpson et al. (1973) reported an increase in lactose concentration in the mammary gland 24 and 48 h after ovariectomy at day 17 of gestation in the rat, but the increased concentration of plasma prolactin was not significantly different from sham-operated (control) rats. On the other hand, Bridges and Goldman (1975) showed that the 446 K. R. Nicholas and P. E. Hartmann increase in prolactin normally observed in late pregnancy was inhibited by ovariectomy. Vermouth and Deis (1974) demonstrated a significant increase in the concentration of prolactin in the plasma 4 h after ovariectomy at day 19 of gestation. The increased levels of prolactin were maintained for at least 58 h and lactogenesis was first detected after 24 h. Progesterone treatment prevented the rise in prolactin observed 4 and 8 h after the removal of the ovaries and delayed the onset of lactogenesis and parturition. The ability of corticosteroids to induce lactogenesis when injected during pregnancy, has been demonstrated in a number of species, including rats (Cowie 1969). Gala and Westphal (l965a) reported a fall in the concentration of corticosteroid binding globulin (CBG) between late pregnancy and early lactation in the rat, and suggested that the increase in biologically active corticosteroid may induce lactogenesis in this species. However, there is still disagreement whether the concentration of corti costeroids in the plasma increases during the final 4-5 days before term in the rat (Kuhn 1969; Denamur 1971; Martin et al. 1977), and thus the involvement of corticosteroids in the lactogenic mechanism is uncertain. In view of the conflicting data concerning the ovarian influence on the increase in prolactin secretion in late pregnancy and the involvement of corticosteroids in the initiation of lactation, the relationship between progesterone, prolactin and corti costeroids during normal lactation and during lactations induced by ovariohysterec tomy and Caesarean section in rats was re-examined. Materials and Methods Animals Female rats (Rattus norvegicus, Wistar strain) were mated overnight, and the detection of sperm on a vaginal smear collected the following morning was taken to indicate day 1 of pregnancy. Rats from the local colony normally give birth early on day 23 of gestation. The animals were provided with food (W. H. Milne and Co., Perth, W.A.) and water ad libitum, and were kept in periods of darkness (2100-0700 hours) and artificial light (0700--2100 hours). Maternal Blood All blood samples were centrifuged at 3000 g for 5 min, and the plasma removed and stored at -15°C until required for analysis. Chronic samples The dorsal aorta was cannulated (Bruce and Cabrol 1975) to obtain blood samples from fully conscious rats. The cannula was exteriorized through a small skin incision over the dorsal neck region. To prevent interference between blood sampling the cannula was temporarily held along the back of the animal in a fold of skin. Just prior to bleeding, an extension (25 cm) of vinyl tube was attached to the cannula and after discarding the first two drops of blood, a sample (0' 1--0· 3 ml) was collected into a heparinized vial. The blood remaining in the cannula and extension tube was returned to the rat with heparinized saline. Rats quickly adapted to this method of sampling and were sampled while walking around the cage, and while eating, sleeping or drinking. Acute samples Blood was collected from the jugular vein of the rat immediately following a blow to its head. Biochemical Analysis Milk was collected from rats and analysed for lactose content as described by Nicholas et al. (1981). The concentration of lactose and glucose in the mammary gland was estimated as previously Plasma Hormones and Lactogenesis in the Rat 447 described (Nicholas and Hartmann 1981a), except that the tissue supernatant was deproteinized before analysis with 5 % (w/v, final concentration) trichloroacetic acid (TCA). This concentration of TCA did not interfere with the standard curves for either lactose or glucose. The concentration of total corticosteroids, CBG capacity, and progesterone in the plasma was determined by the methods of Martin et al. (1977). Plasma levels of prolactin were measured by a double antibody radioimmuno assay with materials and methods supplied by the Rat Pituitary Hormone Program, National Institutes of Arthritis and Metabolic Diseases, Bethesda, Md, U.S.A. The results are expressed in terms of the NIAMD Rat Prolactin-RP-l standard (11 Lu./mg in the mouse deciduoma assay). The sensitivity of the assay was < 0·5 p,g/l. All samples from each experiment were measured in a single assay to eliminate between-assay variation (c. 15 %). Each sample was assayed in duplicate and estimates with a variation > 7 % were not used. 100 .. 80 .. • , A n • 1\ 80~ 60~ 'fb'tP.'h "JI ~ 0..1 i{!' i RI :00 - - 60~ 40~ \. I \ . - II r I... 40 II>c. 1 20t~ ==--0 ot o ,. I ..~ .. I 'c:: 80 400 '" ~ C 0 -0j- c:: , I 1 '0 e Gi II> , 200 'iii 'iii Cl ~ 60 • I 00 II> o .~ ~ c:: c:: 0 o 'f ~ e 40 ~ o 0 ..0 II> U Cl to ... ..:; (5... '"II> . D.. 40 e 20 CI <l III 20 f D.. U • )QJ 0 0 0 . ~ is .. '.'.1 iMI 80 .. E. F 601- \ \ /I V 200 401- I· • .. I ~ . • r0 40 20 20 f 0 0 . 80 -40- -0 80- -40- 0 Time before birth (h) Fig. 1. Progressive changes in the concentrations of progesterone (.), prolactin (1'», corticosteroids (_) and CBG capacity (0) in plasma collected from six rats (A, B, C, D, E, F) over the last 4 days of pregnancy. The rats were cannulated on day 16 of pregnancy and blood sampling commenced on either day 19 or day 20. Blood samples were collected at 12-h intervals for the first 24-36 hand then every 4 h until parturition. The dashed line indicates the time of parturition and the solid bars indicate the hours of darkness. 448 K. R. Nicholas and P. E. Hartmann Results Normal Parturient Rats The time course of the changes in levels of progesterone, prolactin and corticoster oids and CBG capacity in blood samples withdrawn during late pregnancy from six cannulated rats is shown in Fig. 1. Rats C and D gave birth on the morning of d!iy 23, but gestation was 24 h longer in the four remaining animals. The concentratiOJ1 of progesterone in rats C and D declined consistently between 80 and 20 h before parturition, whereas the initial decline in rats A, B, E and F was halted by a transient increase for approximately 20-30 h. In all rats the concentration of progesterone was maintained below 10·0 jlg/l for the final 20 h before parturition. 650 "F :5... CD 550 =.g- 'c: Cl::J 450 ..:!..8 "C'" c:CD ___ 0.