547
J. Physiol. (1972), 253, pp. 547-563 With 7 text-figure8 Printed in Great Britain
THE EFFECTS OF PROLACTIN AND OXYTOCIN ON MILK SECRETION AND ON THE PERMEABILITY OF THE MAMMARY EPITHELIUM IN THE RABBIT
BY J. L. LINZELL, M. PEAKER AND JANET C. TAYLOR* From the Agricultural Research Council, Institnte of Animal Physiology) Babraham, Cambridge CB2 4A T
(Received 13 May 1975) SUMMARY
1. The effects of prolactin or oxytocin on milk secretion and the permeability of the mammary epithelium have been investigated in rabbits. 2. Milk yield was increased by prolactin treatment in late (25-28 days) but not in established (11-14 days) lactation. 3. Prolactin treatment increased milk [lactose] and [K] and decreased [Na] and [Cl] in late lactation, and thus reversed the normal changes in late lactation, but had no significant effect in established lactation. 4. ['4C]sucrose movements from blood to milk were significantly decreased to levels characteristic of established lactation, following prolactin treatment in late lactation. No significant effect was evident with treatment in established lactation. Na and Cl movements showed similar trends. 5. It is suggested that prolactin in some way affects paracellular movements of ions and small molecules like lactose across the mammary epithelium, and that this mechanism is responsible for the changes in the composition of the aqueous phase of milk. 6. Immediately following a single dose of 100 m-u. oxytocin no significant effects on milk composition were evident but after 1 u. milk [Na] and [Cl] were significantly increased. 7. Twenty-four hr after 1 u. oxytocin, milk [Na] and [Cl] were decreased while [K], [lactose], [fat] and [protein] were increased. 8. During an i.v. infusion of oxytocin milk [Na] and [Cl] increased while [K] and [lactose] decreased. The passage of [14C]sucrose, 24Na and 36C0 from blood to milk also increased. 9. These effects of oxytocin are discussed in relation to the permeability *
M.R.C. Scholar.
548 J. L. LINZELL, M. PEAKER AND JANET C. TAYLOR of the mammary epithelium and the pathways for ion movements, and to other studies on milk composition in the rabbit involving the administration of oxytocin to aid in the evacuation of milk. INTRODUCTION
Studies on milk secretion in the rabbit have shown that after about 18 days of lactation, while milk yield remains relatively high, the concentrations of [lactose] and [K] in milk decrease while those of [Na], [Cl], [fat] and [protein] increase (Gachev, 1963a, 1965a, 1971 b; Cowie, 1969; Linzell & Peaker, 1971 a; Peaker & Taylor, 1975). In the previous paper (Peaker & Taylor, 1975) we have investigated the physiological mechanisms responsible for the changes in composition of the aqueous phase of milk in this species and have suggested that throughout lactation there is a paracellular pathway across the mammary epithelium through which ions and small molecules like lactose can pass down their concentration gradients - gradients established by transcellular processes of secretion and ion transport. Evidence was also presented that in late lactation, i.e. when milk [Na] and [Cl] are high and [K] and [lactose] are low, the permeability of the paracellular pathway is increased. The administration of prolactin reverses or prevents the normal changes in milk composition in late lactation (Gachev, 1963a, b, 1965b, 1971a; Cowie, 1969). Therefore it might be argued that prolactin in some way affects the paracellular permeability pathway across the mammary epithelium; the experiments described in this paper, involving permeability measurements in vivo, were designed to test this hypothesis. In view of the wide effects of prolactin in ion and water movements in vertebrate tissues (see Bern & Nicoll, 1968; Nicoll & Bern, 1972) the results may be of wider interest. In most studies on milk secretion in the rabbit, oxytocin has been administered in order to assist in the evacuation of milk (see, for example, Coates, Gregory & Thompson, 1964; Cowie, 1969; Gachev, 1971b, c); in some of these experiments oxytocin was given every day. In ruminants the repeated administration of oxytocin, even in small and seemingly physiological amounts, alters milk composition (see Linzell, 1967; Linzell & Peaker, 1971c). Therefore the question arises of whether the composition of milk obtained after oxytocin administration in rabbits is normal. The change in milk composition induced by oxytocin in goats and cows involves increases in [Na] and [Cl] and decreases in [K] and [lactose] (see Linzell & Peaker, 1971 c). To explain these changes it was shown that, in contrast to the normal situation in lactation, labelled lactose and sucrose cross the mammary epithelium from milk to blood and vice versa, and it
PROLACTIN, OXYTOCIN AND MILK COMPOSITION 549 was suggested that the 'tight junctions' connecting neighbouring secretory cells become 'leaky' following oxytocin treatment, thus permitting ions and small molecules to pass down their concentration gradients between blood and milk. Thus the effects of oxytocin in the rabbit are of interest both in terms of any possible effect on. milk composition and of any increase in epithelial permeability to labelled sucrose. Some of these data have been presented to the Society for Endocrinology (Taylor, Peaker & Linzell, 1975). METHODS
The methods used for studying milk yield, milk composition and the permeability of the mammary epithelium to [14C]sucrose, 24Na and 3 C1 in the rabbit were as described by Peaker & Taylor (1975). Female domestic rabbits of a Dutch strain, in their second to fourth lactation, with four to eight (mean six) young, were used. Prolactin (ovine, NIH-P-S-10, 30 i.u./mg) obtained from the National Institutes of Health, Bethesda, Maryland, U.S.A., was dissolved in 0-154 m-NaCl immediately before subcutaneous injection. The oxytocin was synthetically prepared (Syntocinon: Sandoz). The results obtained with prolactin treatment were compared with data obtained at the same stage of lactation for untreated rabbits by Peaker & Taylor (1975). RESULTS
Prolactin Effects of prolactin in late lactation In seven rabbits prolactin was administered beginning on day 24 of lactation, called day 0 of treatment (25 i.u., twice daily for 2 days, the dose used by Cowie, 1969). Milk yield was measured daily from 6 days before to 7 days after the start of treatment, approximately 1 hr after the morning injection. The animals were milked by hand 12 hr after the last prolactin injection (day 2) in order to study milk composition. The administration of prolactin caused a marked increase in milk yield. This was not evident until day 2 (i.e. 12 hr after the last injection) but the effect persisted for several days, until by day 5 the mean yield was virtually identical to that of the controls (Fig. 1 A). At the peak of the response the yield was increased by approximately 30 %. At the time of maximum yield in the treated group, milk [Na] and [Cl] were significantly lower, and [K] and [lactose] significantly higher than in untreated animals at the same stage of lactation, but not significantly different from animals in established lactation (11-14 days) - the time of minimum milk [Na] and [Cl] and maximum [K] and [lactose] during normal lactation (Fig. 2). Milk [fat] and [protein] showed a tendency to be
550 J. L. LINZELL, M. PEAKER AND JANET C. TA YLOR lower in the prolactin-treated group than in untreated animals at the same stage but the results were not statistically significant (Fig. 2). It has previously been argued that the passage of ['4C]sucrose from blood to milk is a measure of paracellular movements across the mammary epithelium (Linzell & Peaker, 1971 c, 1974; Peaker & Taylor, 1975). A
160 140
120
-
~,L-1
.~1
Prolactin 80
(25 i.u., s.c.)
140
18 19 20 21 22 23 24 25 26 27 28 29 30 31 Days of lactation 0 1 2 3 4 5 6 7 B Time after start of treatment (days)
120
1008
60 60 W Al
I
I
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1(25Prolactin imu., sxc.)
I
I
I
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6 7 8 9 10 11 12 13 14 15 16 17 18 Days of lactation ! 0 1 2 3 4 5 6 7 Time after start of treatment (days) Fig. 1. Effect of prolactin (25 i.u., s.c. twice-daily) on milk yield (closed circles) in rabbits in (A) late lactation (25-28 days) and (B) established lactation (seven animals in each group). Mean ± S.E. of mean. Open circles:
untreated controls.
Therefore the passage of [F4C]sucrose, 24Na and 36C0 from blood to milk was studied in four prolactin-treated rabbits, 12 hr after the last injection. The entry of [14C]sucrose into milk was significantly lower in the treated animals. The mean concentration in milk after 25 min of i.v. infusion was
PROLACTIN, OXYTOCIN AND MILK COMPOSITION 140 K
Na
120
100 80 60
40
P
J. Physiol. (1972), 253, pp. 547-563 With 7 text-figure8 Printed in Great Britain
THE EFFECTS OF PROLACTIN AND OXYTOCIN ON MILK SECRETION AND ON THE PERMEABILITY OF THE MAMMARY EPITHELIUM IN THE RABBIT
BY J. L. LINZELL, M. PEAKER AND JANET C. TAYLOR* From the Agricultural Research Council, Institnte of Animal Physiology) Babraham, Cambridge CB2 4A T
(Received 13 May 1975) SUMMARY
1. The effects of prolactin or oxytocin on milk secretion and the permeability of the mammary epithelium have been investigated in rabbits. 2. Milk yield was increased by prolactin treatment in late (25-28 days) but not in established (11-14 days) lactation. 3. Prolactin treatment increased milk [lactose] and [K] and decreased [Na] and [Cl] in late lactation, and thus reversed the normal changes in late lactation, but had no significant effect in established lactation. 4. ['4C]sucrose movements from blood to milk were significantly decreased to levels characteristic of established lactation, following prolactin treatment in late lactation. No significant effect was evident with treatment in established lactation. Na and Cl movements showed similar trends. 5. It is suggested that prolactin in some way affects paracellular movements of ions and small molecules like lactose across the mammary epithelium, and that this mechanism is responsible for the changes in the composition of the aqueous phase of milk. 6. Immediately following a single dose of 100 m-u. oxytocin no significant effects on milk composition were evident but after 1 u. milk [Na] and [Cl] were significantly increased. 7. Twenty-four hr after 1 u. oxytocin, milk [Na] and [Cl] were decreased while [K], [lactose], [fat] and [protein] were increased. 8. During an i.v. infusion of oxytocin milk [Na] and [Cl] increased while [K] and [lactose] decreased. The passage of [14C]sucrose, 24Na and 36C0 from blood to milk also increased. 9. These effects of oxytocin are discussed in relation to the permeability *
M.R.C. Scholar.
548 J. L. LINZELL, M. PEAKER AND JANET C. TAYLOR of the mammary epithelium and the pathways for ion movements, and to other studies on milk composition in the rabbit involving the administration of oxytocin to aid in the evacuation of milk. INTRODUCTION
Studies on milk secretion in the rabbit have shown that after about 18 days of lactation, while milk yield remains relatively high, the concentrations of [lactose] and [K] in milk decrease while those of [Na], [Cl], [fat] and [protein] increase (Gachev, 1963a, 1965a, 1971 b; Cowie, 1969; Linzell & Peaker, 1971 a; Peaker & Taylor, 1975). In the previous paper (Peaker & Taylor, 1975) we have investigated the physiological mechanisms responsible for the changes in composition of the aqueous phase of milk in this species and have suggested that throughout lactation there is a paracellular pathway across the mammary epithelium through which ions and small molecules like lactose can pass down their concentration gradients - gradients established by transcellular processes of secretion and ion transport. Evidence was also presented that in late lactation, i.e. when milk [Na] and [Cl] are high and [K] and [lactose] are low, the permeability of the paracellular pathway is increased. The administration of prolactin reverses or prevents the normal changes in milk composition in late lactation (Gachev, 1963a, b, 1965b, 1971a; Cowie, 1969). Therefore it might be argued that prolactin in some way affects the paracellular permeability pathway across the mammary epithelium; the experiments described in this paper, involving permeability measurements in vivo, were designed to test this hypothesis. In view of the wide effects of prolactin in ion and water movements in vertebrate tissues (see Bern & Nicoll, 1968; Nicoll & Bern, 1972) the results may be of wider interest. In most studies on milk secretion in the rabbit, oxytocin has been administered in order to assist in the evacuation of milk (see, for example, Coates, Gregory & Thompson, 1964; Cowie, 1969; Gachev, 1971b, c); in some of these experiments oxytocin was given every day. In ruminants the repeated administration of oxytocin, even in small and seemingly physiological amounts, alters milk composition (see Linzell, 1967; Linzell & Peaker, 1971c). Therefore the question arises of whether the composition of milk obtained after oxytocin administration in rabbits is normal. The change in milk composition induced by oxytocin in goats and cows involves increases in [Na] and [Cl] and decreases in [K] and [lactose] (see Linzell & Peaker, 1971 c). To explain these changes it was shown that, in contrast to the normal situation in lactation, labelled lactose and sucrose cross the mammary epithelium from milk to blood and vice versa, and it
PROLACTIN, OXYTOCIN AND MILK COMPOSITION 549 was suggested that the 'tight junctions' connecting neighbouring secretory cells become 'leaky' following oxytocin treatment, thus permitting ions and small molecules to pass down their concentration gradients between blood and milk. Thus the effects of oxytocin in the rabbit are of interest both in terms of any possible effect on. milk composition and of any increase in epithelial permeability to labelled sucrose. Some of these data have been presented to the Society for Endocrinology (Taylor, Peaker & Linzell, 1975). METHODS
The methods used for studying milk yield, milk composition and the permeability of the mammary epithelium to [14C]sucrose, 24Na and 3 C1 in the rabbit were as described by Peaker & Taylor (1975). Female domestic rabbits of a Dutch strain, in their second to fourth lactation, with four to eight (mean six) young, were used. Prolactin (ovine, NIH-P-S-10, 30 i.u./mg) obtained from the National Institutes of Health, Bethesda, Maryland, U.S.A., was dissolved in 0-154 m-NaCl immediately before subcutaneous injection. The oxytocin was synthetically prepared (Syntocinon: Sandoz). The results obtained with prolactin treatment were compared with data obtained at the same stage of lactation for untreated rabbits by Peaker & Taylor (1975). RESULTS
Prolactin Effects of prolactin in late lactation In seven rabbits prolactin was administered beginning on day 24 of lactation, called day 0 of treatment (25 i.u., twice daily for 2 days, the dose used by Cowie, 1969). Milk yield was measured daily from 6 days before to 7 days after the start of treatment, approximately 1 hr after the morning injection. The animals were milked by hand 12 hr after the last prolactin injection (day 2) in order to study milk composition. The administration of prolactin caused a marked increase in milk yield. This was not evident until day 2 (i.e. 12 hr after the last injection) but the effect persisted for several days, until by day 5 the mean yield was virtually identical to that of the controls (Fig. 1 A). At the peak of the response the yield was increased by approximately 30 %. At the time of maximum yield in the treated group, milk [Na] and [Cl] were significantly lower, and [K] and [lactose] significantly higher than in untreated animals at the same stage of lactation, but not significantly different from animals in established lactation (11-14 days) - the time of minimum milk [Na] and [Cl] and maximum [K] and [lactose] during normal lactation (Fig. 2). Milk [fat] and [protein] showed a tendency to be
550 J. L. LINZELL, M. PEAKER AND JANET C. TA YLOR lower in the prolactin-treated group than in untreated animals at the same stage but the results were not statistically significant (Fig. 2). It has previously been argued that the passage of ['4C]sucrose from blood to milk is a measure of paracellular movements across the mammary epithelium (Linzell & Peaker, 1971 c, 1974; Peaker & Taylor, 1975). A
160 140
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Prolactin 80
(25 i.u., s.c.)
140
18 19 20 21 22 23 24 25 26 27 28 29 30 31 Days of lactation 0 1 2 3 4 5 6 7 B Time after start of treatment (days)
120
1008
60 60 W Al
I
I
I
I
1(25Prolactin imu., sxc.)
I
I
I
I
I
5
6 7 8 9 10 11 12 13 14 15 16 17 18 Days of lactation ! 0 1 2 3 4 5 6 7 Time after start of treatment (days) Fig. 1. Effect of prolactin (25 i.u., s.c. twice-daily) on milk yield (closed circles) in rabbits in (A) late lactation (25-28 days) and (B) established lactation (seven animals in each group). Mean ± S.E. of mean. Open circles:
untreated controls.
Therefore the passage of [F4C]sucrose, 24Na and 36C0 from blood to milk was studied in four prolactin-treated rabbits, 12 hr after the last injection. The entry of [14C]sucrose into milk was significantly lower in the treated animals. The mean concentration in milk after 25 min of i.v. infusion was
PROLACTIN, OXYTOCIN AND MILK COMPOSITION 140 K
Na
120
100 80 60
40
P
