Suckling-Induced Serum Prolactin Levels Are Modified by Interference with Milk Ejection in Lactating Rats¹

^a Instituto de Neurobiología, Buenos Aires, Argentina ^b Laboratorio de Investigaciones en Neuroendocrinología de la Reproducción Humana (LINERH), Hospital Carlos G. Durand, Buenos Aires, Argentina

	ABSTRACT

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The aim of this study was to determine whether suckling-induced prolactin (PRL) levels were modified when milk ejection was impaired. Milk ejection impairment was achieved in two experimental models: a) depriving the dam of sleep during suckling and b) increasing the nonsuckling intervals in lactating dams.

Sleep deprivation blocked milk ejection and enhanced suckling-induced PRL levels in dams that had been previously separated from their pups. When milk ejection is blocked in litter-deprived dams, mammary glands are not evacuated and they remain engorged. Suckling stimuli were not the cause of the difference in suckling-induced serum PRL levels in control and sleep-deprived dams.

The engorgement of the mammary glands may play a role, as a maximum suckling-induced PRL increase was not observed in nonseparated SD dams with nonengorged mammary glands. Moreover, suckling-induced PRL levels were decreased when engorged mammary glands of SD dams were evacuated through an oxytocin injection.

A parallel increase between suckling-induced PRL levels and mammary gland weight was observed in the experiments in which milk ejection was impaired through an increase in the intervals of nonsuckling, providing additional support for a relationship between mammary gland engorgement and the regulation of suckling-induced PRL levels.

	INTRODUCTION

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Litters get necessary nourishment through suckling, thus producing hormonal changes in the mother for the production of milk. Suckling is not a simple phenomenon. It comprises a series of oral movements having two main functions: 1) to stimulate nipple receptors that originate afferent nerve potentials resulting in the release of prolactin (PRL) and oxytocin (Oxy) in the mother and 2) to draw out milk from the cistern and large ducts. Therefore, lactation is a twofold process that includes 1) milk production by mammary glands, with PRL involved as the primary hormone, and 2) transfer of milk from the mammary glands to the offspring, with Oxy and the oral movements of the infant being the factors that allow successful transference.

It is generally accepted that in dams, serum PRL levels are dependent on the week of lactation, on the number of pups, and on the period of time pups are attached to the nipples. One of the models most often used to study suckling-induced PRL release includes separating pups from dams during a certain period of time and then replacing litters and allowing them to suckle again [1–4]. Obviously, if lactating dams are litter deprived, the milk is not ejected, and as a result the mammary glands are not evacuated. When pups are replaced and allowed to suckle again, serum PRL levels begin to increase and then continue to increase during the suckling period, showing high values. Ten to fifteen minutes after the beginning of the suckling bout, the milk is ejected and mammary glands are emptied [5–8].

However, higher serum PRL levels are not observed in dams that were never litter deprived [9, 10], suggesting that other factors besides suckling are involved in enhanced suckling-induced serum PRL levels.

At present, it is not known whether the impairment of milk ejection is involved in suckling-induced hyperprolactinemia. The aim of this study was to study suckling-induced serum PRL levels in two experimental models. In both models, milk ejection was impaired. Groups consisted of 1) lactating dams that were deprived of sleep while suckling and 2) lactating dams that were litter deprived for 4 and 16 h, after which litters were replaced and allowed to suckle again.

	MATERIALS AND METHODS

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Holtzman lactating dams on day 10 of lactation were used throughout the study period. Purina (Corgill-Purina, Buenos Aires, Argentina) rat chow and water were administered ad libitum. The animals were housed under controlled temperature and light conditions (lights-on from 0600 to 2000 h).

About 2–3 days after parturition, the dam and the litter were placed in an individual cage in an isolated testing room. Eight pups for each dam were left in the cage. Special care was taken to avoid noises or other disturbances.

Separated and Nonseparated Dams

Separated dams were those from which litters were removed for a period of time. Nonseparated dams were always kept together with their litters.

Separated and Nonseparated Litters

Separated litters were those separated from dams during 4 h prior to the experiment. Nonseparated litters were always kept together with a dam.

Sleep-Deprived and Control Dams

Sleep-deprived (SD) dams were animals that were experimentally prevented from sleeping while suckling according to a procedure previously described. The milk ejection reflex (MER) is triggered by suckling only when the dam falls asleep—not when she is awake. Once mothers crouch over pups and pups become attached, lactating dams gradually fall asleep and an intermittent milk ejection takes place [5]. Sleep was impaired in SD mothers by keeping them alert during the suckling period. Sleep impairment was achieved by showing a pencil and moving it or by using other mild stimuli such as touching the whiskers softly with a pencil or gently blowing on them. As dams got used to the stimuli and consequently fell asleep, alternate stimuli were used to keep them alert. In SD lactating rats, milk ejection did not take place. Control (C) dams were allowed to sleep while being suckled, and milk was normally ejected.

Measurement of the Suckling Period

After placement of litters in the cage together with dams, the onset of suckling bouts began after a variable period of time, depending mainly on the mother's willingness to nurse and on the pups' willingness to suckle. Dams gathered pups and crouched over them. The suckling period in all experimental setups started when dams adopted an arched position with pups attached. The suckling period represents the actual time pups were attached to the nipples.

Experiments with Impairment of Milk Ejection through Dams' Sleep Deprivation

Experiment 1 Two groups of dams were used: SD and C dams. Dams were litter deprived for 4 h. Then pups were replaced and allowed to suckle for 30 min.

Experiment 2 After having been separated for 4 h, pups were replaced and allowed to suckle during different time periods (5, 10, 15, 20, or 30 min). One group included C dams and the other SD dams.

Experiment 3 Separated and nonseparated pups were allowed to suckle both C and SD lactating dams for 30 min. Dams that had been separated from their pups for 4 h were used.

Experiment 4 Nonseparated dams and 4 h-separated pups were used. One group included SD dams and the other C dams. Both groups suckled for 30 min.

Experiment 5 The effect of sleep deprivation in lactating dams that were not allowed to be suckled was studied during 30 min. Dams separated from their pups during 4 h were used.

Experiment 6 The effect of an Oxy injection during suckling on SD lactating dams was studied. The right jugular vein was cannulated through the right atrium 48 h before experimentation. On the day the experiment took place, dams were litter deprived for 4 h. Pups were then replaced and allowed to suckle for 30 min while dams were deprived of sleep. Every 10 and 20 min after the beginning of the suckling period, dams received an Oxy injection (0.5 IU; Sandoz, Basel, Switzerland). Another group injected with saline was used as control.

Experiment 7 This experiment was performed in order to test whether engorged or nonengorged mammary glands modify suckling-induced serum PRL levels in SD dams. Mothers separated from their pups for 4 h and nonseparated mothers were used. The same suckling pups, separated for 4 h, were used for both experimental groups. Both groups were suckled for 30 min.

Experiments with Impairment of Milk Ejection through Periods of Nonsuckling in Lactating Dams

Experiment 8 Dams separated from their pups (for 4 or 16 h) and nonseparated lactating dams were used. One group of separated dams was suckled for 30 min and the other was not. In order for the suckling stimulus to remain constant, pups separated from their mothers for 4 h were used.

Experiment with Lactating Dams Suckled by Separated or Nonseparated Litters

Experiment 9 Lactating dams separated for 4 h from their litters were used. They were allowed to be suckled by their own 4 h-separated pups or by nonseparated pups. Litters were allowed to suckle during 5, 15, or 30 min.

At the end of all the experiments, animals were killed by decapitation. Trunk blood was collected and mammary glands were dissected out and weighed. Milk from the pups' stomachs was pressed away and weighed. In experiment 9 (using separated and nonseparated pups), the milk content was not measured because the stomachs of nonseparated pups were full of milk from a previous ingestion. Instead, in both groups the milk intake could be determined by weighing the pup's body before and after the suckling period. It should be noticed that the differences in pups' weights reflected not only the milk intake but also weight loss due to urine excretion during suckling. The number of stretch reactions during suckling was also registered in experiment 9.

Macrophotographs of dissected right abdominal mammary glands were obtained. Scanning microscopic observations were carried out using an Olympus OVM 1000NM (Tokyo, Japan) video microscope attached to a Sony Mavigraph VP 3000 (Atsugi, Japan) videoprinter.

PRL RIA

Blood samples for PRL measurements were allowed to clot at room temperature for 3 h. The serum was separated by centrifugation and frozen at -20°C until assayed. PRL was measured by double-antibody RIA with material supplied by the NIDDK program (NIH, Bethesda, MD). Reference preparations were rat RP3. Data were expressed as means ± SEM.

Statistical Analysis

Statistical analysis was performed with Student's t-test in all sleep deprivation experiments, as each SD group had its own control group. Student's t-test was also performed in experiment 9, in which the suckling stimulus from 4 h-separated and nonseparated pups was compared. In the experiment using lactating dams with different nonsuckling periods in which more than two groups were compared (experiment 8), ANOVA was performed and differences among groups were determined by the Newman-Keuls test.

	RESULTS

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Suckling-Induced Serum PRL Levels after Impairment of Milk Ejection through Sleep Deprivation

SD lactating dams that were litter deprived for 4 h ejected less milk and showed a higher serum PRL level than C dams (Fig. 1a). Figure 1b shows the amount of milk suckled by litters and the serum PRL levels after various suckling periods in both C and SD dams. About 10 min after suckling, the MER started in the control group. After this period, the quantity of milk found in litters of SD animals was lower than in those of the C group because the MER took place only in C animals. It is to be highlighted that when the MER is not triggered, the quantity of milk found in the pups' stomach is the remainder from a previous ingestion. Five minutes after suckling, serum PRL values were increased in both the C and SD groups. During the first 10 min, no difference was observed between the C and SD groups. Fifteen minutes after suckling, lactating SD dams showed a higher serum PRL level than the C group. The difference observed in serum PRL levels between SD and C animals was significant through to the end of the experiment.

View larger version (17K): [in this window] [in a new window]   FIG. 1. a) Amount of milk obtained by pups and the suckling-induced serum PRL levels in litter-deprived C dams and in litter- and sleep-deprived dams (SD). Pups were allowed to suckle for 30 min. Data are expressed as means ± SEM. ***p < 0.001. Numbers of dams: C, 22; SD, 17. b) Amount of milk (bars) obtained from pups and serum PRL levels (circles) in litter-deprived C dams and in litter- and sleep-deprived dams (SD). Pups were allowed to suckle for 5, 10, 15, 20, and 30 min. Data are expressed as means ± SEM. The number of animals ranged between 8 and 11. About 10 minutes after suckling, the MER begins. *p < 0.03; **p < 0.04; ***p < 0.001.

Figure 2a shows results for C and SD separated dams suckled either by separated or by nonseparated litters. The mammary glands were heavier and serum PRL levels higher in SD than in C dams suckled by separated or nonseparated pups. Significant differences in suckling-induced serum PRL levels or in mammary gland weight between SD and C groups were not observed when dams had not been previously separated and suckled by separated litters (Fig. 2b). Sleep deprivation for 30 min did not affect basal serum PRL levels in dams separated from their litters for 4 h (Fig. 2c).

View larger version (17K): [in this window] [in a new window]   FIG. 2. a) Mammary gland weight and suckling-induced serum PRL levels in C and SD separated lactating dams that were suckled by separated or nonseparated pups. Suckling occurred for 30 min. Data are expressed as means ± SEM. The number of animals used per group ranged from 7 to 11. *p < 0.01; **p < 0.02; ***p < 0.001. b) Mammary gland weight and serum PRL levels in C and SD nonseparated dams suckled by 4 h-separated pups. Suckling occurred for 30 min. Data are expressed as means ± SEM. Number of animals ranged from 7 to 10. c) Serum PRL levels in litter-deprived C and in litter and 30 min-sleep-deprived (SD) dams that were not allowed to be suckled. Number of animals ranged from 7 to 10. Data are expressed as means ± SEM.

Figure 3a shows the effect of the evacuation of engorged mammary glands of separated SD dams on serum PRL levels. The evacuation of glands was produced by an Oxy injection during suckling. The Oxy injection caused the milk let down and lowered the weight of mammary glands and suckling-induced serum PRL levels.

View larger version (18K): [in this window] [in a new window]   FIG. 3. a) Suckling-induced serum PRL levels and mammary gland and milk weight in SD lactating dams injected with saline (sep-saline) or Oxy (sep-oxy). All animals were separated for 4 h and then allowed to be suckled for 30 min and deprived of sleep. Injections were given at 10 and 20 min after the beginning of the suckling period. Number of animals ranged from 9 to 11. Data are expressed as means ± SEM. *p < 0.01; **p < 0.002; ****p < 0.0001. b) Suckling-induced serum PRL levels and mammary gland and milk weight in SD lactating dams previously separated for 4 h (sep) or not separated (non-sep) and allowed to be suckled, with sleep deprivation, for 30 min. Both groups were suckled by litters previously separated from their mothers for 4 h. Number of animals was between 8 and 11. Data are expressed as means ± SEM. ***p < 0.001.

The effect of engorged or nonengorged mammary glands of SD dams on suckling-induced serum PRL levels is shown in Figure 3b. Both groups of SD dams had the same suckling stimulus from 4 h-separated pups. Suckling-induced serum PRL levels were higher in SD dams with engorged (heavier) mammary glands than in SD dams with nonengorged mammary glands.

Mammary Gland, Acini, and Alveoli Size

Figure 4 shows that the mammary glands, acini, and alveoli of nonseparated lactating dams (Fig. 4a) were smaller than those of separated dams (4 h) that were milk engorged (Fig. 4b). The mammary glands of a separated dam were emptied 30 min after suckling, and they were similar in size as those of a nonseparated dam, with acini and alveoli that were also similar (Fig. 4c). Conversely, 30 min after suckling, mammary glands were not evacuated in SD dams. The mammary gland remained engorged, and the sizes of the mammary gland, acini, and alveoli were also similar to those in separated dams (Fig. 4d).

View larger version (46K): [in this window] [in a new window]   FIG. 4. Photographs and microphotographs (insets) of the right abdominal mammary gland of lactating dams. a) Nonseparated: the dam remained with the litter until animals were killed. b) Separated: the mother was separated from the litter for 4 h and then killed. c) Separated control: the mother was separated from the litter for 4 h, then allowed to be suckled without any disturbances for 30 min, and killed. d) Separated, sleep-deprived (SD): the mother was separated from the litter for 4 h, then allowed to be suckled for 30 min, with sleep deprivation, and then killed. The line at bottom right indicates 2 cm for photographs and 1 mm for microphotographs.

Suckling-Induced Serum PRL Levels in Lactating Dams after Impairment of Milk Ejection through Various Nonsuckling Periods

Figure 5a shows serum PRL values of nonseparated (0 h) and separated (4–16 h) dams. Dams separated for 4–16 h were divided into two groups: one was allowed to be suckled and the other was not. Suckling-induced serum PRL levels were increased when the nonsuckling period was longer, but levels were diminished in dams that were not suckled. Mammary gland weight in nonsuckled dams was increased when the nonsuckling period was longer (Fig. 5b).

View larger version (17K): [in this window] [in a new window]   FIG. 5. a) Serum PRL levels in lactating dams not allowed or allowed to be suckled for 30 min; nonseparated or separated for 4 and 16 h. Number of animals was between 9 and 15. Data are expressed as means ± SEM. ***p < 0.001 vs. 0 h; **p < 0.01 vs. 0 h; *p < 0.05 vs. 0 h. b) Mammary gland weight of lactating dams not allowed to be suckled for 0 (nonseparated) or 4 or 16 h. Number of animals was between 9 and 15. Data are expressed as means ± SEM. ***p < 0.001 vs. 4 and 16 h; **p < 0.01 vs. 16 h.

Suckling-Induced Serum PRL Levels in Lactating Separated Dams Suckled by Nonseparated or Separated Litters for Different Periods of Time

Figure 6 shows the results from comparison of the suckling stimuli from separated and nonseparated litters on lactating dams suckled for 30 min or for shorter periods of time. Mammary gland weight, pup weight differences, serum PRL levels, and the number of stretch reactions are shown for separated dams suckled by separated or nonseparated pups for 5, 15, and 30 min. There were no differences between separated and nonseparated groups of pups in any of the parameters studied.

View larger version (28K): [in this window] [in a new window]   FIG. 6. a) Mammary gland weight of litter-deprived dams suckled by separated or nonseparated pups during 5, 15, or 30 min. Number of animals was between 8 and 12. Data are expressed as means ± SEM. b) Body weight difference of separated and nonseparated pups after suckling litter-deprived dams for 5, 15, or 30 min. Number of animals was between 8 and 12. Data are expressed as means ± SEM. c) Serum PRL levels in litter-deprived dams suckled by separated or nonseparated pups during 5, 15, or 30 min. Number of animals was between 8 and 12. Data are expressed as means ± SEM. d) The number of stretch reactions in litters after suckling of litter-deprived dams for 5, 15, or 30 min. Separated and nonseparated pups were used. Number of animals was between 8 and 12. Data are expressed as means ± SEM.

	DISCUSSION

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Our results show that the impairment of milk ejection, produced either by sleep deprivation in separated dams while suckling or by an increase in nonsuckling periods in lactating dams, resulted in heavier mammary glands and higher suckling-induced serum PRL levels.

When milk ejection is impaired through sleep deprivation in previously separated dams, suckling-induced serum PRL levels increase markedly. The MER is initiated in C animals at 10–15 min after the beginning of the suckling period, but not in SD dams [5, 6]. From then on, PRL levels in SD animals were higher than in the C group. The relationship between the absence of the MER and the increase in serum PRL levels in SD dams suggests a linkage between the ejection of milk and serum PRL levels.

There are two main points to be considered when milk ejection is blocked: pups do not receive nourishment, and mammary glands remain engorged. Pups used in our model were deprived of food for 4 h. When they were placed with their mothers again, pups suckling the C dams obtained milk, filled their stomachs, and became gradually satiated, whereas pups suckling the SD dams did not obtain milk, their stomachs remained empty, and they were not satiated. Satiated and nonsatiated pups may produce different suckling stimuli, and it is therefore conceivable that this factor could be responsible for the differences in serum PRL levels between C and SD dams. Experiment 3 shows that in the case of nonseparated pups with full stomachs, the differences in serum PRL levels between C and SD dams were still observed. These results rule out the possibility that stimuli from nonsatiated pups with empty stomachs are the cause of the maximum increase of serum PRL levels in SD separated dams. Moreover, the suckling stimulus does not cause higher PRL levels in SD dams with nonengorged mammary glands (nonseparated SD dams). These two facts suggest that engorgement of the mammary glands rather than the suckling stimuli may be the cause of the increased serum PRL levels in SD dams.

It is known that separated pups ingest more milk than nonseparated pups if it is sufficiently supplied by dams that have been separated from their pups at least for 8 h or when they suckle for longer periods (4 h or longer) [11, 12]. Conversely, our results show that separated pups ingest the same amount of milk as nonseparated pups when they suckle a 4 h-separated dam and for up to 30 min. Moreover, changes in serum PRL levels were not observed in the case of pups deprived of food for 4 h. This suggests that 4-h food deprivation is not enough stimulus to produce changes in PRL levels; such changes have been reported by Terkel [12], who used chronically underfed pups that had been deprived of food for 24 h.

It has been reported that afferent nerve impulses evoked by suckling are different before and during milk ingestion [13]. Therefore, one can speculate that because milk ejection is blocked in SD dams, afferent nerve potentials could be different in these animals and could modify PRL release. However, we found that the same dry suckling stimuli resulted in higher suckling-induced serum PRL levels only in separated SD dams, not in nonseparated SD rats. The results rule out the possibility that dry suckling is the cause of higher suckling-induced serum PRL levels observed in SD dams.

A maximum suckling-induced PRL increase was observed only in sleep- and litter-deprived dams whose mammary glands remained engorged as sleep deprivation impaired evacuation of the stored milk. When dams were not litter deprived (there was no mammary engorgement), milk impairment in SD dams did not increase suckling-induced PRL levels, supporting the existence of a relationship between mammary engorgement and enhanced serum PRL levels. Moreover, when engorged mammary glands of SD dams were emptied by Oxy during suckling, lower serum PRL levels were observed, suggesting that mammary gland engorgement could exert a control action on suckling-induced serum PRL levels.

In nonseparated lactating dams whose mammary glands are not engorged because of constant milking activity, only low serum PRL values are observed [9, 10]. We carried out experiments both with dams that were not litter deprived and with dams separated from their pups during 4 and 16 h, and we found the highest serum PRL levels in mothers with 16-h separation. The maximum suckling-induced serum PRL levels observed after 16 h of separation may be produced by a higher release of PRL due to an increase of pituitary stores. However, an additional involvement of mammary gland engorgement cannot be disregarded, as these experiments show a parallel increase between mammary gland weight and serum PRL levels.

It has been reported that Oxy has a modulatory action on PRL secretion [14–17]. In our experiments with SD dams, it was assumed that Oxy was not released as milk ejection was blocked in these animals; therefore the higher PRL levels observed in SD dams cannot be attributed to endogenous Oxy. However, in the experiment using Oxy injections in SD dams, serum PRL levels decreased. Similar results were obtained by Yamamuro and Sensui [18], who reported that an Oxy injection lowered suckling-induced serum PRL levels. Thus, the modulatory mechanisms of Oxy on serum PRL levels may be attributed to an action on PRL release and/or indirectly to the evacuation of the mammary glands.

Sleep deprivation and mammary gland engorgement may generate some discomfort in the mother. Lactating dams do not react against stressful situations as nonlactating dams do [19, 20]. While in nonlactating dams the stress causes a serum PRL increase, it is widely known that stress in lactating dams produces either a decrease in serum PRL levels or no changes at all. Our results show that basal PRL levels (without suckling) are not modified by sleep deprivation or by engorgement of the mammary glands. Thus, the maximum increase in suckling-induced serum PRL levels observed after sleep deprivation cannot be attributed to stress.

The ways in which mammary gland engorgement might enhance suckling-induced PRL levels are as follows. First, neural or humoral [21] signals from engorged mammary glands could modify hypothalamic or hypophysial mechanisms involved in the regulation of PRL release. Second, mammary glands take up PRL from blood [22, 23]; but when they are engorged, this mechanism could be interrupted. Studies in cows [24] show that an evacuated mammary gland incorporates more PRL in milk than a nonevacuated gland, supporting our point of view that there may be a relationship between serum PRL levels and mammary gland evacuation.

	ACKNOWLEDGMENTS

 
The authors wish to thank Marcela Huerta, Patricia Louzan, Ignacio Fossati, and Silvina Heisecke for their assistance and Dr. Parlow (NIDKK program) for providing RIA materials. We are also grateful to Miriam Golía for her assistance in the English version of the paper.

	FOOTNOTES

 
¹ This work has been supported by the Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET) and by Fundacion Instituto de Neurobiologia, Buenos Aires, Argentina.

² Correspondence: Serrano 669, (1414) Buenos Aires, Argentina. FAX: 54–1–856–7108; fuacta{at}ssdnet.com.ar

Accepted: March 3, 1998.

Received: January 21, 1997.

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