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 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.. ~ ~ ·0 ~ 80 r 60 .. 1 ',_ 350 Q; CD "'- ...... o 0 '"o '"0 , 60 Cl ~ too )C~! c: .+:; 40 =l~ 0 T ~ 0 0 19 20 21 22 Days pregnant Fig. 2. The concentrations of progesterone (.,), prolactin (6), corticosteroids (_) and CBG activity (D) in the plasmaofrats duHng the final 4 days of pregnancy. Each value represents the mean ± s.e.m. for four or five observations. The concentration of prolactin in the plasma remained at 5-10 jlg/l from 100 h before parturition until the concentration of progesterone fell to < 10·0 jlg/l. In this connection, rat F presents an interesting case, since a transient increase in the concentration of prolactin (to 14 jlg/l) occurred 44 h before term, after the first decline in progesterone (7·0 jlg/l), but prolactin then decreased when the concentration of progesterone increased to 20 jlg/l. A second increase in prolactin concentration (to 42 ltg/I) was observed 20 h before parturition following the decrease in plasma progesterone from 20·0 to 3 jlg/l. The concentration of prolactin in the plasma from all cannulated rats increased to 20-90 jlg/l prior to term. Although the concentration of corticosteroids in the plasma ranged from 50 to 450 jlg/l, no consistent circadian rhythm was demonstrated. The concentration of corticosteroids and CBG capacity in the plasma was determined during late pregnancy in rats A, B, C and D, and ranged from 50 to 450 jlg/l and from 40 to 350 jlg corti- Plasma Hormones and Lactogenesis in the Rat 449 costerone bound per litre, respectively. However, both the CBG capacity and the concentration of corticosteroids within each rat remained relatively constant during late pregnancy. 80 (a) 60 100 ,R. , \ 40 , \ 80 , \ _-I:) \ / , \ f---__~------/, \ \ _s-- / ------.:-c::a_--- 60 I _---"\/ _---- A ~f------t~------20 "\ p-, II _---, \ 40 """ \ //'~'/ ---- / \ / .... ~\ / I ,\ _-----" ...... , .... \X , I ''0..__-::.-=:::::_ ----- __-----_--v 20 Cl '/~ ~/1-.j.__ ~ ----- CD C e 0 T -1 I , ,0 CD 0: 4 16 20 24 ...II> CD :::::::.-' Cl Cl 0 ~ Ii: (b) /l 80 'i', / 160 .~ / )" co I / /,, " , / " £ , I I ~ '--..?, I II // ',', 120 I 60· II ,// ..... ,', V d" '~ I ',.,A I / ' Fig. 3. Progressive changes in the plasma concentrations of progesterone (solid lines) and prolactin (dashed lines) during the first 24 h after surgery for either six ovariohysterectomized rats (a) or six Caesarean-sectioned rats (b), on day 19 of pregnancy. All rats were cannulated on day 16 of pregnancy and bleeding commenced just prior to either ovariohysterectomy or Caesarian section. The respective progesterone and prolactin values for individual rats are represented by matching symbols. 450 K. R. Nicholas and P. E. Hartmann While all cannulated rats did not appear to have a difficult labour and the litter size was between 6 and 14 pups, rat C died 12 h after parturition and rats E and F suckled their respective litters for less than 24 h. Rats A, Band D suckled litters for at least 5 days. The mean concentrations of progesterone, prolactin, corticosteroids and CBG capacity in the plasma from rats killed at intervals of 24 h for 4 days prepartum and at parturition are presented in Fig. 2. The decline in progesterone concentrations between 48 and 24 h before term coincided with increases in the levels of prolactin which continued to increase until parturition. The concentrations of corticosteroids (250-550 fJg/l) and CBG capacities (250-480 fJg corticosterone bound per litre) re mained relatively constant. 3·0 ""0 ~ c > '" ""0 0,1: 2 2·5 >01 ~.Q) 0) Fig. 4. Mammary gland weight (_) E s: 8 2·0 and the concentration of lactose (.) E N -...., and glucose (.a.) in the mammary tissue :2'" E! 1·5 of rats for 48 h after either 5 ovariohysterectomy (dashed lines) or Caesarean section (solid lines) on day Q) :l 4 19 of pregnancy. Each value represents the mean ± s.e.m. for four or five '';::;'" Ol 3 observations. "- Ol .s 2 /" / Q) / B'" u O.4 Q) /" :l ...J'" Q) '" o .~ • j0·2 '" '" 0 u Ol : :~~ ~" o (!)... .s01 o 12 24 36 48 Time after surgery (h) Ovariohysterectomized and Caesarean-sectioned Rats The concentrations of progesterone and prolactin in the plasma collected from cannulated rats after ovariohysterectomy at day 19 of gestation are shown in Fig. 3a. Progesterone concentrations declined abruptly from 40-80 fJg/l to less than 20 fJg/l within 30 min of ovariohysterectomy and thereafter remained less than 10·0 fJg/1. The concentrations of prolactin were constant at approximately 5 fJg/l for 4-8 h after removal of the ovaries and uterus, and then increased gradually during the subsequent 16-18 h In contrast, the concentrations of progesterone in the plasma collected from cannulated rats after Caesarean section at day 19 of gestation declined gradually from 40-80 fJg/l to less than 10·0 fJg/l within 10-14 h (Fig. 3b). The concentrations of prolactin in the same samples remained constant at 5-10 fJg/l for at least 6 h, and then increased markedly between 12 and 16 h after surgery. The concentrations of progesterone (> 45 fJg/l) and prolactin « 10 fJg/l) in the plasma of three sham-operated rats remained constant for 24 h after sham surgery. c ______• ______ Plasma Hormones and Lactogenesis in the Rat 451 The concentrations of lactose and glucose in the mammary tissue, and the wet weights of the inguinal mammary gland during the 48 h after Caesarean section and ovariohysterectomy are presented in Fig. 4. Ovariohysterectomy was followed by a gradual increase in the concentrations of mammary lactose during the first 18 h, and then a further marked increase to a maximum concentration of 3· 26 mg/g after 36 h. The wet weight of tissue was maximal (2'3 g per 200 g body wt) 24 h after surgery. Lactose was first detected (0'14 mg/g) in the mammary gland 12 h after Caesarean section and concentrations subsequently increased from 0·23 mg/g at 18 h to 0·65 mg/g at 24 h. Thereafter the concentration of lactose increased sevenfold to reach its maximum of 4· 58 mg/g at 48 h. The concentration of glucose in the mammary gland increased from 0·09 mg/g at day 19 of gestation to 0·25 mg/g at 36 h after removal of the ovaries and/or foetoplacental unit. Table 1. Milk lactose concentration and milk content of the mammary gland of rats after either Caesarean section or ovariohysterectomy on day 19 of gestation Milk samples for the determination of lactose concentration were collected at 24, 36 and 48 h after surgery. The milk content of the mammary gland was calculated from the concentration of lactose in the milk and in the mammary tissue. Each value represents the mean ± s.e.m. for four or five observations Time after Caesarean section Ovariohysterectomy surgery (h) Milk lactose Gland milk Milk lactose Gland milk (g/lOO ml) (ml/gland) (g/100 ml) (ml/gland) 24 1·61±O·27 O·O6±O·01 1·95±O·1l O·14±O·Ol 36 2·84±O·O3 O·24±O·O4 2·28±O·13 O'25±O'O4 48 2·65±O·18 O·41±O·O4 1·87±O·15 O·14±O·01 The concentrations of lactose in the mammary secretion and the milk content of the mammary gland at 24, 36 and 48 h after Caesarean section and ovariohysterectomy are presented in Table 1. The concentrations of milk lactose were not significantly different between groups after 24 h. However, the milk content of the glands of ovariohysterectomized rats was higher. The concentrations of lactose in the milk increased to reach maximum levels 36 h after both treatments, but the milk contents of the gland continued to increase up to 48 h only in Caesarean-sectioned rats. Discussion Since the concentration of prolactin and corticosteroids is greatly affected by stress (see Beigelman et al. 1956; Lawson and Gala 1974) it was important to use non stressful methods of sampling in the present study. In rats the best method appears to be decapitation, without anaesthesia, within seconds of removal of the animal from the cage (Lawson and Gala 1974, 1975; Hawkins et al. 1975; Dohler et al. 1976). However, this limits the interpretation of the data and increases the number of animals required for a particular study. The effects of surgical stress, blood loss and anaes thetics on plasma prolactin concentration in ovariectomized rats with indwelling aortic cannulae have been considered by Lawson and Gala (1974). They concluded that recovery from ovariectomy and cannulation was complete by 3 days after the operation. Smith and Neill (1976) reported that surgical placement of cannulae at least 2 days before the initial breeding permitted expression of both diurnal and noc- 452 K. R. Nicholas and P. E. Hartmann turnal prolactin surges during the first half of pregnancy in the rat. In addition, Lawson and Gala (1974) demonstrated a significant decline in the concentration of plasma prolactin 20 min after the removal of I . 2 ml of blood with no saline replace ment. Thus in the present study, blood samples were obtained from rats either by stunning and immediate decapitation, or from indwelling cannulae The cannulae were inserted 3 days before the initial blood sampling, and the sample volume was limited to less than O· 3 ml with saline replacement. Moreover, except for the first three or four samples which were obtained within 1 h after ovariohysterectomy (sample volume O· 1 ml), the interval between sampling was at least 2 h. The decline in the concentrations of progesterone and the corresponding increase in prolactin levels found in the plasma collected from rats killed over intervals of 24 h in late pregnancy (Fig. 2), are in agreement with previous reports (Amenomori et al. 1970; Denamur 1971; Bast and Melampy 1972; Linkie and Niswender 1972; Morishinge et al. 1973; Nicholas and Hartmann 1981a) and with the changes observed in chronically cannulated rats (Fig. 1). These findings confirm the conclusions of Bartholomeus et al. (1976) that parturition occurs approximately 24 h after the con centration of progesterone has declined to less than 12·0 f1gjl. In four of the six chronically cannulated rats there was a transient halt to the initial decline in plasma progesterone between 40 and 50 h before birth, and then progesterone declined to low levels and birth occurred approximately 24 h later. It is of interest that birth was delayed by 24 h in all of these four rats. The concentration of progesterone also decreased progressively over a period of 10-14 h in rats Caesarean-sectioned on day 19 of gestation. This slow decline in progesterone probably reflects the half-life of the placental luteotrophic hormone. An abrupt increase in plasma prolactin levels occurred when the progesterone con centrations had declined to less than 10· 0 f1gjl, suggesting that the stimulus for this increase in prolactin release in late pregnancy in the Caesarean-sectioned rat was related to the decline in plasma progesterone to low levels. The oestrogen to progesterone ratio increases in the plasma of the rat during late pregnancy (Soloff et al. 1979). This increase is due to an increase in the concentration of oestrogen (Y oshinaga et al. 1969; Shaikh 1971) and to a decrease in the concen tration of progesterone (Soloff et al. 1979). Administration of oestrogen to rats after the removal of the ovaries increases prolactin secretion (Chen and Meites 1970; Lawson and Gala 1974; Vermouth and Deis 1974), while the administration of progesterone immediately after ovariectomy prevents this increase (Vermouth and Deis 1974). In the present study the abrupt increase in concentrations of plasma prolactin following withdrawal of progesterone after a Caesarean section contrasts with the delayed and gradual increase following withdrawal of progesterone after ovariohysterectomy (Fig. 3); this supports the idea that the ovary is involved in the mammary secretory processes (Bridges and Goldman 1975). Lactose was detected in the mammary gland 12 h after Caesarean section and ovariohysterectomy (Fig. 4). However, the rate of accumulation of lactose was greater between 12 and 24 h after surgery in the ovariohysterectomized rats (Fig. 4) (cf. Kuhn 1969). These changes in gland lactose were closely related to the fall in plasma pro gesterone and the rise in prolactin. The maximum concentration of lactose in the mammary tissue, together with the milk content of the mammary gland, was greater after Caesarean section than after ovariohysterectomy (Table 1). This may be a consequence of a higher prolactin concentration in the plasma after Caesarean section Plasma Hormones and Lactogenesis in the Rat 453 since the administration of ovine prolactin to rats after ovariohysterectomy increased the concentration of lactose in the mammary gland (Nicholas and Hartmann 198Ib). After comparing CBG values measured on the third day before birth with those measured on the third day of lactation, Gala and Westphal (I965a) suggested that a marked decline in CBG would increase the level of unbound corticosteroids (con sidered to be the active form of the hormone) required to initiate lactation. However, in the present, more intensive study of the changes in CBG capacity during the last 4 days of pregnancy (Fig. 1), only small changes in CBG were observed at the time of lactogenesis. The concentration of corticosteroids and the CBG capacity of the plasma during late pregnancy in cannulated rats (Fig. 3) were lower than those observed in daily samples taken from decapitated rats (Fig. 2). These lower levels of corticosteroids and CBG, plus the failure of the rats to sustain elevated levels of prolactin after the withdrawal of progesterone in late pregnancy, may be attributed to a decline in blood volume (see Lawson and Gala 1974). The abrupt changes in CBG capacity in several rats which occurred in the 4-h interval between successive samples reflects a rapid turn over of this binding protein. The binding capacity has been shown to increase in response to the administration of thyroxine and progesterone (Gala and Westphal 1965b, 1966a), and is depressed by glucocorticoids and testosterone (Gala and Westphal 1965b, 1966b; Seal and Doe 1965). However, the acute changes in CBG capacity did not appear to be related to fluctuations in the concentrations of either progesterone or corticosteroids (Fig. 1). Taken collectively, the present findings support the hypothesis that progesterone withdrawal is the lactogenic signal (Kuhn 1969) and suggest the involvement of prolactin. An increase in the concentration of free corticosteroids in late pregnancy with a related stimulatory role in the lactogenic mechanism was not established. Acknowledgments We thank Jane Wilson and Helen Nottage for their skilled technical assistance. The work was supported by the National Health and Medical Research Council of Australia. References Amenomori, Y., Chen, C. L., and Meites, J. (1970). Serum prolactin levels in rats during different reproductive states. Endocrinology 86, 505-10. Bartholomeus, R. K., Bruce, N. W., Martin, C. E., and Hartmann, P. E. (1976). Serial measurement of arterial plasma progesterone levels throughout gestation and parturition in individual rats. Acta Endocrinol. (Copenhagen) 82, 436-43. Bast, J. D., and Melampy, R. M. (1972). Luteinizing hormone, prolactin and ovarian 20tX-hydroxy steroid dehydrogenase levels during pregnancy and pseudopregnancy in the rat. Endocrinology 91, 1499-505. Beigelman, P. M., Slusher, M. A., Slater, G. G., and Roberts, S. (1956). Effects of anaesthetics and collection time on corticosteroid secretion by rat adrenal. Proc. Soc. Exp. BioI. Med. 93, 608-11. Bridges, R. S., and Goldman, B. D. (1975). Ovarian control of prolactin secretion during late pregnancy in the rat. Endocrinology 97, 496-8. Bruce, N. W., and Cabral, D. (1975). Effects of maternal blood loss on embryonic and placental development in the rat. J. Reprod. Ferfil. 45, 349-56. Chen, C. L., and Meites, J. (1970). Effects of oestrogen and progesterone on serum and pituitary prolactin levels in ovariectomised rats. Endocrinology 86, 503. 454 K. R. Nicholas and P. E. Hartmann Cowie, A. T. (1969). General hormonal factors involved in lactogenesis. Tn 'Lactogenesis: The Initiation of Milk Secretion at Parturition'. pp. 157-69. (Univ. Pennsylvania Press: Pennsyl vania.) Denamur, R. (1971). Hormonal control of lactogenesis. J. Dairy Res. 38, 237-64. Dohler, K. D., von zur Miihlen, A., Gartner, K., and Dohler, V. (1976). Various blood sampling techniques and their effects on pituitary hormones in rats. Acta Endocrinol. (Copenhagen) Suppl. 202,29-30. Gala, R. R., and Westphal, V. (1965a). Corticosteroid-binding globulin in the rat: possible role in the initiation of lactation. Endocrinology 76, 1079-88. Gala, R. R., and Westphal, V. (1965b). Corticosteroid-binding globulin in the rat: studies on the sex difference. Endocrinology 77, 841-51. Gala, R. R., and Westphal, V. (1966a). Influence of anterior pituitary hormones on the corticosteroid binding globulin in the rat. Endocrinology 79, 55-66. Gala, R. R., and Westphal, V. (1966b). Further studies on the corticosteroid-binding globulin in the rat: Proposed endocrine control. Endocrinology 79, 67-76. Hawkins, R. A., Freedman, B., Marshall, A., and Killen, E. (1975). Oestradiol-17jJ and prolactin levels in rat peripheral plasma. Br. J. Cancer 32, 179-85. Kuhn, N. J. (1969). Progesterone withdrawal as the lactogenic trigger in the rat. J. Endocrinol. 44, 39-54. Lawson, D. M., and Gala, R. R. (1974). The influence of surgery, time of day, blood volume reduction and anaesthetics on plasma prolactin in ovariectomized rats. J. Endocrinol. 62, 75-83. Lawson, D. M., and Gala, R. R. (1975). Influence of anaesthetics on basal, perphenazine-induced and thyrotrophin releasing hormone-induced prolactin secretion in ovariectomized, oestrogen treated rats. J. Endocrinol. 66, 151-7. Linkie, D. M., and Niswender, G. D. (1972). Serum levels of prolactin, luteinizing hormone, and follicle stimulating hormone during pregnancy in the rat. Endocrinology 90, 632-7. Martin, C. E., Cake, M. H., Hartmann, P. E., and Cook, I. F. (1977). Relationship between foetal corticosteroids, maternal progesterone and parturition in the rat. Acta Endocrinol.. (Copenhagen) 84,167-76. Morishinge, W. K., Pepe, G. J., and Rothchild, I. (1973). Serum luteinizing hormone, prolactin and progesterone levels during pregnancy in the rat. Endocrinology 92, 1527-30. Nicholas, K. R., and Hartmann, P. E. (1981a). Progesterone control of the initiation of lactose synthesis in the rat. Aust. J. Bioi. Sci. 34, 435-43. Nicholas, K. R., and Hartmann, P. E. (1981b). The foetoplacental unit and the initiation oflactation in the rat. Aust. J. Bioi. Sci. 34, 455-61. Nicholas, K. R., Hartmann, P. E., and McDonald, B. L. (1981). IX-Lactalbumin and lactose concen tration in rat milk during lactation. Biochem. J. 194, 149-54. Seal, U. S., and Doe, R. P. (1965). Verterbrate distribution of corticosteroid-binding globulin and some endocrine effects of concentration. Steroids 5, 827-41. Shaikh, A. A. (1971). Estrone and estradiol levels in the ovarian venous blood from rats during the estrous cycle and pregnancy. Bioi. Reprod. 5, 297-307. Simpson, A. A., Simpson, N. H. W., and Kulkarni, P. N. (1973). Prolactin production and lacto genesis in rats after ovariectomy in late pregnancy. J. Endocrinol. 57, 425-9. Smith, M. S., and Neill, J. D. (1976). Termination at midpregnancy of the two daily surges of plasma prolactin initiated by mating in the rat. Endocrinology 98, 696-701. Soloff, M. S., Alexandrova, M., and Fernstrom, M. J. (1979). Oxytocin receptors: triggers for parturition and lactation. Science 204, 1313-15. Vermouth, N. T., and Deis, R. P. (1974). Prolactin release and lactogenesis after ovariectomy in pregnant rats: effect of ovarian hormones. J. Endocrinol. 63, 13-20. Yoshinaga, K., Hawkins, R. A., and Stocker, J. F. (1969). Estrogen secretion by the rat ovary in vivo during the estrous cycle and pregnancy. Endocrinology 85, 103-12. Manuscript received 15 September 1980, accepted 4 May 1981