The prolactin concentration in the plasma of lactating rats rose less rapidly and attained a significantly lower plateau level in response to suckling on day 20–21 of lactation than it did on day 13–14 of lactation. Neither differences in suckling stimulation of the older pups nor a higher metabolic clearance rate (MCR) of prolactin were implicated in the reduced prolactin concentration seen in the late-lactating rats. The MCR was, in fact, slightly reduced in both conscious and late-lactating rats anaesthetized with urethane when compared with those in mid-lactation. The MCR of prolactin was not significantly altered by urethane anaesthesia in rats on either day of lactation. However, the secretion rate of prolactin, computed from the MCR multiplied by the equilibrum concentration of prolactin during suckling, was considerably reduced (665 to 392 ng/min) from mid- to late lactation. We conclude from these data that the reduced plasma concentration of prolactin in response to suckling in late lactation is the result of an impairment within the prolactin secretory mechanism.

SUMMARY

Rat prolactin (11 i.u./mg) was continuously infused into the circulation of urethaneanaesthetized lactating rats for 35 min at doses of either 200 or 472 ng/min. The immunoreactive prolactin in both milk and plasma rose quickly during the first 20–25 min of infusion, then stabilized at similar levels over baseline (68 and 98 ng/ml for milk and plasma, respectively, with the 200 ng/min dose and 250 and 230 ng/ml, respectively, with the 472 ng/min dose). The concentration of prolactin in plasma fell after the infusion was stopped, whereas that in the milk either did not fall at all or fell slightly to a new stabilized level. There was a rapid and extensive loss in the immunoreactivity of prolactin added to milk when rat milk was incubated in vitro (37 °C for 1–120 min) with either 600 ng/ml of extracted pituitary prolactin (NIAMDD RP-1) or unit equivalent amounts of prolactin obtained from pituitary culture media (secreted prolactin, supplied by C. S. Nicoll). Significantly greater amounts of added RP-1 prolactin were lost when it was incubated with milk obtained after 4 h than after 18 h of non-suckling. There was, however, no drop in endogenous immunoreactive milk prolactin levels (350–400 ng/ml) when rat milk was incubated with saline for 30 min. This suggests that milk prolactin obtained as a result of plasma transfer is different chemically from the milk prolactin resulting from the addition of either RP-1 or secreted prolactin to milk in vitro.

Approximately 90% of ¹³¹I-labelled rat prolactin appeared in the trichloroacetic acid precipitable fraction after incubation (37 °C for 120 min) with milk obtained after 4 h of non-suckling in either the presence or absence of thiouracil (added to prevent binding of ¹³¹I or ¹³¹I-labelled fragments to milk protein). The recovery was slightly less when ¹³¹I-labelled prolactin was incubated with milk obtained after 18 h of non-suckling.

These data suggest that prolactin is quickly transferred from plasma into milk in direct relation to the plasma concentration. Once there, much of it appears to be retained by the milk perhaps chemically or physically bound; there is little, if any, degradation of the hormone. We conclude that the lactating rat mammary gland may function normally as an excretory organ for prolactin.

SUMMARY

Milk was added intraductally in 0·2 ml increments to previously emptied mammary glands in rats. Direct microscopic observations of the glands showed that each increment of milk did not distribute evenly either within a single lobule or among lobules. Increase of the intramammary pressure either by i.v. injection of oxytocin or by compression of the gland between thumb and forefinger, distributed the milk more evenly within the gland. These observations led to the routine injection of 1·6–3·2 mu. oxytocin in order to distribute each increment of milk evenly throughout the gland. The intramammary pressure response of the gland to different doses of oxytocin was then determined. The rises in intramammary pressure above equilibrium pressure in response to 0·8 and 1·6 mu. oxytocin increased fourfold on average with increasing gland volume over the compliant portion of the static pressure-volume curve. The slopes of the dose oxytocin-intramammary pressure response lines became progressively steeper and shifted to the left as the volume of the mammary gland increased, indicating a progressive reduction in the dose of oxytocin required to elicit a given response. No differences were noted between rats lactating for 9–13 days and those lactating for 20–24 days. In a related study the duct system of the rat gland was found to contain little fluid even after 8 h of non-suckling, though it became filled swiftly after a single i.v. injection of oxytocin. Non-suckling for 16–24 h was required to overcome passively the resistance to entry of milk from the alveoli.

SUMMARY

The mammary glands of rats either maintained continuously in the animal room on day 14 post partum or isolated in a room without other rats on day 21 post partum, refilled only partially within 8 h after the pups had withdrawn the milk from the glands by suckling with the aid of oxytocin injections to the mother. The partial refilling occurred regardless of whether or not the release of prolactin at the time of suckling was blocked by an injection of an extract of stalk median eminence (SME). The mammary glands of rats not injected with SME continued to refill and reached near capacity within 16 h whereas those of the SME-injected rats failed to refill any further. The glands of the SME-injected rats could be stimulated to refill more completely within 8 h after the initial emptying provided prolactin either was injected or was released endogenously by exposing the mothers to exteroceptive signals from their pups midway during the 8-h period of non-suckling which preceded the initial emptying. More complete mammary refilling also occurred within 8 h if a 4-h rather than an 8-h period of non-suckling preceded the initial emptying of the glands. The results of these investigations indicate that the prolactin released by suckling at the time of the initial emptying of the glands influenced only that milk which reaccumulated in the emptied glands during the 8th—16th h following suckling. The milk which reaccumulated during the first 8 h appeared to be influenced by prolactin released several hours before suckling.

The results suggest, therefore, that a time delay of several hours exists from the time of release of prolactin by suckling until the full stimulatory effect of the hormone upon milk secretion is manifest. Thus the milk secreted after emptying of the glands at a given suckling would be the result of prolactin released earlier either at previous sucklings or in response to exteroceptive stimulation.

SUMMARY

The results of experiments in which the prolactin in the primiparous rat pituitary was bioassayed suggested that the failure of suckling to release prolactin after 8 h of non-suckling on day 21 post-partum was due to the fact that prolactin had been discharged from the pituitary during the 8-h non-suckling period, presumably by exteroceptive signals emanating from the general environment of the animal room. This was substantiated in other experiments in which prolactin release was assessed indirectly through its stimulatory effects upon milk secretion. In these experiments, the mammary glands of rats maintained continuously in the animal room filled faster on day 21 after complete emptying of the glands by exogenous oxytocin, than did either rats on day 14 post-partum maintained continuously in the animal room or rats isolated in a room without other rats on day 21 post-partum. The glands of the latter two groups of rats could be stimulated to fill faster provided prolactin was injected 4 h before the initial emptying of the glands. The exteroceptive stimuli in the animal room environment that stimulated the release of prolactin in the 21-day post-partum rat apparently emanated at least in part from other lactating rats and/or their litters, since faster mammary gland refilling occurred in isolated 21 day post-partum rats when they were exposed to the presence of lactating rats with their litters for 30 min halfway through the 8-h non-suckling period which preceded the initial emptying of the gland. Exposure to male rats, on the other hand, was totally ineffective. A release of prolactin occurred in response to animal room environmental stimuli in the day 14 primiparous rat provided 13–14 day old foster pups were inserted in place of the mother's own pups on day 7. Thus, the rapidly changing characteristics of the pups from 14 to 21 days of age in some manner is involved in the increasing responsiveness of the exteroceptive mechanism for prolactin release which occurs from day 14 to day 21 post-partum.

Lactating rats were fed at a level equivalent to 75% of the daily food consumed by controls, which had food available ad libitum, from days 4–14 or from days 4–12 post partum. The underfed mothers lost about 10% of their initial weight whereas control mothers gained weight slightly during this time. The litters (six pups) of underfed mothers gained 20% less weight than those of controls but remained healthy. The milk obtained by the pups from six glands (the remaining six glands had been ligated on day 3) during a timed suckling, plus that subsequently obtained after oxytocin injections to the mother, totalled 7·4± 0·80 g in underfed and 15± 0·62 g in control mothers on day 14, and 6 ± 0·41 g in underfed compared with 8 ±0·50 g in control mothers on day 12. The pups of underfed mothers obtained a significantly greater percentage of the total milk after 10 (30 v. 6%), 15 (84 v. 32%) and 45 (100 v. 57%) min of suckling than did those of control mothers. In two trials for the milk ejection test, the litters of control and underfed mothers were exchanged. The pups of control mothers suckled by underfed mothers in both trials obtained a significantly greater percentage of the total milk (64%) in 15 min than did the pups of underfed mothers suckled by control mothers (25%).

Oxytocin-induced intramammary pressure (IMP) responses recorded under urethane anaesthesia achieved a greater amplitude in underfed than in control mothers in response to each dose (0·4, 0·8 and 1·6 mu.) of oxytocin administered intravenously. The average slope of the dose–response curve was 1·25 cm H₂O/0·1 mu. oxytocin compared with 0·75cm H₂O/0·1 mu. for fed mothers. The amplitude of the IMP in response to a 1·6 mu. dose of oxytocin also was greater in underfed mothers over a range of volumes of milk added incrementally to empty glands. The difference was most striking at low volumes of milk (0·2–0·6 ml) within the gland. The data from these experiments suggest the more rapid transfer of milk from mother to pups in underfed rats is due at least in part to adaptations within the mammary gland, possibly involving reductions in the sympathetic-mediated tone of mammary ducts.

Prolactin passes from the systemic circulation of lactating rats into the milk where it can be consumed by the young rats during suckling. ¹³¹I-Labelled rat prolactin was detected in the plasma of 9- to 14-day-old rats after being nursed by mothers previously injected with ¹³¹I-labelled rat prolactin and after the pups had received ¹³¹I-labelled rat prolactin by gastric intubation. It was estimated that 16% of the ¹³¹I-labelled rat prolactin given by gastric intubation subsequently appeared in the plasma of the neonate. Gastric administration of 10·5 or 21·0 μg B-1 rat prolactin significantly raised the level of prolactin in the plasma of 13-day-old pups, but a similar increase was not observed when 27-day-old rats were given 46·2 μg B-1 prolactin by gastric intubation. The concentration of prolactin in the plasma of 13- to 14-day-old rats rose to 55 ng/ml 30 min after the onset of nursing by mothers whose mammary glands were full of milk, whereas the concentration in the plasma of offspring suckled by mothers with empty mammary glands remained at basal values. It is concluded that the intestine of the newborn rat is permeable to prolactin and that milk may constitute an exogenous source of prolactin for the suckled offspring.

ABSTRACT

Adenohypophysial prolactin of lactating rats was pulse-labelled by [³H]leucine injected i.v. at the time of removal of the pups. The [³H]prolactin concentration in the pituitary gland, analysed by polyacrylamide-gel electrophoresis, progressively fell as the time from labelling to removal of the pituitary gland increased from 8 to 24 h, which suggests that there was a loss of hormone as it aged within the gland. Suckling effectively provoked the depletion–transformation of total and [³H]prolactin (extracted at pH 7·2) when applied after 8 h but not when applied after either 16 or 24 h after removing the pups.

In rats whose pups were removed for 8 h, suckling also depleted–transformed [³H]prolactin labelled 4 h, but not that labelled 1 h before suckling. The pituitary glands of other lactating rats were labelled with [³H]leucine injected i.v. at various times before removing the glands and incubating them in medium 199. The secretion into the medium of [³H]prolactin labelled either 4, 8, 16 or 24 h beforehand was maximal during the first 30 min then declined from 30 to 240 min of incubation. However, secretion of prolactin labelled 1 h and 10 min beforehand reached a maximum after 0·5–1 h and 2 h of incubation respectively, then remained constant during the remainder of the 4-h incubation period. The total 4-h secretion of [³H]prolactin was greatest (65% of preincubation concentration) from those glands labelled 4 h before in contrast to those labelled 10 min (15%) or 1 (38%), 8 (34%), 16 (18%) or 24 h (26%) before incubation. Taken together, these data suggest that prolactin synthesized 4 h earlier is more likely to be released in response to physiological stimuli than is more recently formed prolactin or prolactin which has remained in the pituitary gland for 16 h or longer.

J. Endocr. (1984) 101, 27–32

SUMMARY

Milk yield fell in a single rabbit mammary gland within 24 h after the litter had been reduced from six to eight pups to one pup. The diminished secretion lasted 2–3 days, then rebounded to the expected level over the next 1–2 days and thereafter maintained this level. During early to mid-lactation the decrease in milk secretion after reduction of the litter to one pup was prevented either by s.c. injections of prolactin or by s.c. injections of the β-adrenergic blocking drug, propranolol. These results suggest that milk secretion was reduced in the single gland as the result of sympathetic activation, triggered possibly by the turgid state which developed in the remaining glands after the litter was reduced. The results suggest also that the depression of milk secretion by the sympathetic system was due to a reduction in the effective amount of prolactin and probably of other adenohypophysial hormones reaching the mammary secretory cells rather than to an impairment of their release from the anterior pituitary.

The magnitude of the depression and rebound in milk secretion of a single rabbit mammary gland, and the effectiveness of the preventive action of either propranolol or prolactin, was considerably less during late as compared with early to mid-lactation. We postulate from these results that the mammary gland of the rabbit has a functional lability, i.e. responsiveness, which diminishes as lactation progresses and which dictates the extent to which local, neural and humoral factors may alter the milk secretory function of the mammary gland. Since suckling in the rabbit does not change from a once-a-day frequency throughout lactation, a diminishing lability may be an important factor in the decline and cessation of lactation in this species.

SUMMARY

The metabolic clearance of rat prolactin, assessed by the continuous infusion to equilibrium method, increased from 0·77 to 2·15 ml/min in lactating and from 0·65 to 1·75 ml/min in non-lactating rats as the infusion rate was increased from 100 to 472 ng/min. The clearance rate of rat prolactin did not increase when large doses of ovine prolactin were infused simutaneously with rat prolactin. The clearance rates computed from equilibrium levels of immunoreactive ¹³¹I-labelled rat prolactin in plasma were highly correlated with, but were considerably greater than, those computed from unlabelled rat prolactin which was infused simultaneously. In lactating and non-lactating rats, the clearance of each prolactin tested (RP-1, B-1, Nicoll's secreted and ¹³¹I-labelled) increased and had stabilized within the 30–35 min of infusion of each new higher dose of the hormone. The ability of the rat to clear quickly greater amounts of prolactin from the circulation as the infusion rate increases implies that plasma prolactin concentrations do not necessarily mirror the rate of prolactin secretion from the pituitary.