Neuroendocrinology of maternal behavior in the rabbit

Rabbit maternal behavior consists of building an underground nest of straw and body hair during late pregnancy and displaying, with circadian periodicity, a single 3-min nursing bout/day across lactation. Estrogen, androgen, progesterone, and prolactin regulate specific aspects of nest-building and promote the onset of maternal responsiveness. However, the maintenance of this behavior relies on stimuli from the litter: by preventing mother/young contact at parturition or during early lactation maternal responsiveness is altered or abolished. The brain areas controlling the expression of nest-building and nursing were investigated by implanting estradiol, locating the distribution of estrogen and prolactin receptors, quantifying the expression of immediate-early genes, and lesioning structures of the olfactory system. These studies revealed that: (a) estrogen receptor-alpha, alpha, present in the preoptic region, may mediate the stimulation of nest-building by estradiol; (b) prolactin binding sites, located mainly in periventricular structures, are more abundant in late pregnancy and early lactation; (c) the number of FOS-immunoreactive neurons increases in the lateral septum, but not in the mediobasal hypothalamus, following nursing; (d) the accessory olfactory bulb tonically inhibits the expression of maternal behavior because its removal promotes maternal responsiveness in virgins, which are otherwise unresponsive to daily pup exposure. In summary, rabbits rely on the same hormonal and extrahormonal factors that stimulate maternal behavior in other mammals, yet the way in which such factors promote elaborate nest-building and the unfailing display of circadian nursing is unique to rabbits and warrants future investigation.     

Aspects of the biochemistry, physiology and endocrinology of lactation

Recent research on the endocrine control of the initiation of lactation, on the hypothalamic control of prolactin secretion, and on the utilization of nutrients by the mammary gland for the synthesis of milk is reviewed. Particular emphasis is placed on the mechanism of action of prolactin, and the role played by prolactin receptors.     

Regulation of ghrelin secretion during pregnancy and lactation in the rat: possible involvement of hypothalamus

We investigated the plasma concentration of ghrelin peptide during pregnancy and lactation in rats. Plasma ghrelin levels on days 10 and 15 of pregnancy were significantly lower than those of the non-pregnant rats. Thereafter, the plasma ghrelin levels on day 20 of pregnancy sharply increased to levels comparable with those in non-pregnant rats. Ghrelin peptide concentrations in the stomach did not change significantly during pregnancy. In the hypothalamus, ghrelin mRNA levels were significantly lower on day 15 of pregnancy than in the non-pregnant rats. Also, plasma ghrelin levels were significantly lower in lactating dams than non-lactating controls on days 3 and 8 of lactation. We examined the possible involvement of prolactin and oxytocin in the regulation of plasma ghrelin concentrations during lactation. Although plasma prolactin levels were decreased by the administration of bromocriptine, plasma ghrelin levels did not differ significantly between vehicle- and drug-treated lactating rats. Administration of haloperidol produced a marked increase in plasma prolactin levels as compared with the non-lactating controls. However, plasma ghrelin levels were not significantly different between vehicle- and drug-treated rats. Administration of an oxytocin antagonist into the lateral ventricle significantly inhibited the increase in the plasma oxytocin level induced by acute suckling. However, plasma ghrelin levels did not significantly between the groups. These observations indicated that the decrease in serum ghrelin is caused by a loss of the contribution of hypothalamic ghrelin. Furthermore, the present results suggested that the suckling stimulus itself, but the release of prolactin or oxytocin, is the factor most likely to be responsible for the suppression of ghrelin secretion during lactation.     

Prolactin inhibition in lactating rats changes leptin transfer through the milk.

Malnutrition during lactation reduces milk production and changes pup's leptin serum levels. To test prolactin role in this nutritional state, we evaluated whether prolactin suppression during lactation changes serum leptin in dams, its transfer through the milk, and pup's serum leptin. Lactating rats were treated with bromocryptine (1 mg/twice a day, s.c.) or saline three days before sacrifice (days 2-4 or days 19-21). Food intake and body weight were measured until sacrifice (4th and 21st day). Serum prolactin and leptin were determined by radioimmunoassay. Bromocryptine injected dams had lower serum prolactin and milk production as expected. The mothers presented lower food ingestion (day 21: -25%), lower body weight (day 4: -12%; day 21: -10%), higher serum leptin (day 4: +68%), lower milk leptin on the 4th day (11 times) and higher (8 times) on the 21st day. The offspring of bromocryptine-treated mothers presented lower body weight in both periods of lactation and lower serum leptin on the 4th day (-40%) and higher on the 21st day (+37%) of lactation. We suggest that prolactin, through its effect on leptin secretion into the milk, may play an important role in signalizing maternal nutritional status to the pups.     

Neuroendocrine actions and regulation of hypothalamic neuropeptide Y during lactation

The expression of neuropeptide Y (NPY) and its co-messenger, agouti-related peptide (AgRP), in arcuate neurons of the hypothalamus is increased during lactation in rats. Our research has been addressing the questions of the physiological actions of these peptides during lactation and the physiological signals associated with lactation that result in increased expression of their genes. Our studies indicate that NPY and AgRP exert pleiotropic actions during lactation that help integrate neuroendocrine regulation of energy balance with controls over anterior and posterior pituitary hormone secretion. Further, reciprocal signaling to the NPY/AgRP system by leptin and ghrelin is responsible for the changes in expression of these hypothalamic peptides in lactating animals, and thus, may contribute to regulation of food intake and the various neuroendocrine adaptations of lactation.     

Male pheromones initiate prolactin-induced neurogenesis and advance maternal behavior in female mice

Prolactin is required for rapid onset of maternal behavior after parturition, inducing adaptive changes in the maternal brain including enhanced neurogenesis in the subventricular zone during pregnancy. The resultant increase in olfactory interneurons may be required for altered processing of olfactory cues during the establishment of maternal behavior. Pheromones act through olfactory pathways to exert powerful effects on behavior in rodents and also affect prolactin secretion. Hence, this study aimed to investigate the effect of male pheromones on neurogenesis and maternal behavior in female mice. Virgin female mice were housed individually or in split-cages where they had pheromonal but not physical contact with a male. Maternal behavior was assessed in a foster pup retrieval paradigm. Some mice were injected with bromodeoxyuridine, and the labeled cells visualized using immunohistochemistry. The data show that exposure to male pheromones, for a duration equivalent to a murine pregnancy, advanced maternal behavior in both virgin and postpartum female mice. The pheromone action was dependent on prolactin and ovarian steroids, and was associated with increased cell proliferation in the subventricular zone and subsequent increases in new neurons in the olfactory bulb. Moreover, the effect of pheromones on both cell proliferation and maternal behavior could be induced solely through administration of exogenous prolactin to mimic the pheromone-induced changes in prolactin secretion. The data suggest that male pheromones induce a prolactin-mediated increase in neurogenesis in female mice, resulting in advanced maternal behavior.     

Effects of ergocryptine on prolactin secretion druing concurrent pregnancy and lactation in the rat

Ergocryptine (2 mg/kg) caused short- and long-term reduction of prolactin secretion in rats experiencing concurrent lactation and pregnancy. The long-term effects of the drug lasted at least 60 days and resulted in reduced milk secretion and termination of pregnancy. Prolactin replacement therapy at a low dose (5 i.u./day) was unsuccessful in overcoming these effects but a higher dose (up to 60 i.u./day) increased milk production and maintained pregnancy. One possible explanation of these results is that prolactin, rather than the suckling stimulus, was responsible for the suppression of oestrous cycles, because ergocryptine brought about a resumption of oestrous vaginal smears in all treated rats in spite of continued suckling.     

Maternal nurturing is dependent on her innate anxiety: the behavioral roles of brain oxytocin and vasopressin

The maternal brain undergoes remarkable physiological and behavioral changes in the peripartum period to meet the demands of the offspring. Here, the brain neuropeptides oxytocin and vasopressin, together with prolactin, play important roles. These neuropeptides are critically involved in the regulation of maternal behavior. Furthermore, reduced anxiety in lactation is another adaptation of the maternal brain. Therefore, a link between maternal behavior and maternal anxiety has been repeatedly postulated. This is supported by our studies in rats bred for high (HAB) and low (LAB) anxiety-related behavior. While female HAB rats become less anxious in lactation, their anxiety level is still four times higher compared with LAB dams. Interestingly, HAB dams display an intense and protective mothering style including increased arched back nursing and pup retrieval whereas LAB dams display only low levels of maternal care. The amount of maternal care directed towards the pups correlates with the mother's innate anxiety. In addition to differences in maternal care, HAB dams are also more protective as they show heightened aggression against a virgin intruder compared with the less aggressive LAB dams. The level of maternal aggression correlates with both their innate anxiety level as well as with the release of oxytocin and vasopressin in hypothalamic and limbic brain areas. Importantly, manipulations of the brain oxytocin and vasopressin systems alter maternal behavior and — depending on the brain region — can also alter the dam's anxiety. Thus, the mother's innate anxiety determines her maternal performance and oxytocin and vasopressin are involved in both parameters.     

Maternal behavior in pigs

When sows kept under commercial conditions were put into crates in the early 1960s, the neuro-endocrine regulation of the maternal behavior in these domestic animals was disputed. Thus, the study of sow maternal behavior intensified and today a significant body of knowledge has accumulated to support the hormonal regulation of sow maternal behavior. The onset of nest building is associated with a periparturient decline in progesterone, an increase in prolactin and a major rise in plasma concentrations of PGF2alpha the day before parturition. Some nest building behaviors, such as pawing and gathering straw, have been found to correlate with changes in the levels of progesterone, prolactin and somatostatin. The duration of the birth process correlates negatively with peripheral oxytocin levels. During lactation, the stimuli from the piglets affect the release of several hormones which not only regulate the let down of milk but also sow metabolism and mammary milk production. The sow's nursing behavior ensures an even distribution of milk to her piglets. The piglets suckling behavior, in turn, is mainly a way to communicate their individual nutritional needs.     

Changes of sex hormone-binding globulin/SHBG expression in the hypothalamo-hypophyseal system of rats during pregnancy, parturition and lactation.

Sex hormone-binding globulin (SHBG) is expressed in hypothalamic magnocellular neurons. High co-localization rates of SHBG with oxytocin have been observed in the hypothalamus, indicating that SHBG plays a role in pregnancy, parturition and lactation. Further studies have shown that hypothalamic SHBG expression is malleable to changing steroid conditions. In this study, we have examined SHBG levels in the supraoptic and paraventricular hypothalamic nuclei and in the posterior pituitary lobe of late pregnant, parturient and early lactating rats by IN SITU hybridization, immunocytochemistry, and ELISA. Immunocytochemical and biochemical analysis showed that the SHBG levels increased during late pregnancy in hypothalamic nuclei. During parturition, SHBG levels fell in the magnocellular nuclei but increased in the posterior pituitary lobe. SHBG levels increase again during lactation. At day six of lactation, there was no significant difference in SHBG levels compared to normal cycling female rats, which served as control in this study. IN SITU hybridization showed increased SHBG mRNA signal during late pregnancy. The highest SHBG expression was observed during parturition. Our data indicate that hypothalamic SHBG expression changes during pregnancy, parturition and lactation, parallel to ovarian steroid and co-localized OT levels. This may in part be linked to known steroid actions on synthesis and secretion of magnocellular hypothalamic peptide hormones, important for the control of parturition and lactation.     

The expectant brain: adapting for motherhood

A successful pregnancy requires multiple adaptations of the mother's physiology to optimize fetal growth and development, to protect the fetus from adverse programming, to provide impetus for timely parturition and to ensure that adequate maternal care is provided after parturition. Many of these adaptations are organized by the mother's brain, predominantly through changes in neuroendocrine systems, and these changes are primarily driven by the hormones of pregnancy. By contrast, adaptations in the mother's brain during lactation are maintained by external stimuli from the young. The changes in pregnancy are not necessarily innocuous: they may predispose the mother to post-partum mood disorders.     

Reciprocity in the Developmental Regulation of Aquaporins 1, 3 and 5 during Pregnancy and Lactation in the Rat

Milk secretion involves significant flux of water, driven largely by synthesis of lactose within the Golgi apparatus. It has not been determined whether this flux is simply a passive consequence of the osmotic potential between cytosol and Golgi, or whether it involves regulated flow. Aquaporins (AQPs) are membrane water channels that regulate water flux. AQP1, AQP3 and AQP5 have previously been detected in mammary tissue, but evidence of developmental regulation (altered expression according to the developmental and physiological state of the mammary gland) is lacking and their cellular/subcellular location is not well understood. In this paper we present evidence of developmental regulation of all three of these AQPs. Further, there was evidence of reciprocity since expression of the rather abundant AQP3 and less abundant AQP1 increased significantly from pregnancy into lactation, whereas expression of the least abundant AQP5 decreased. It would be tempting to suggest that AQP3 and AQP1 are involved in the secretion of water into milk. Paradoxically, however, it was AQP5 that demonstrated most evidence of expression located at the apical (secretory) membrane. The possibility is discussed that AQP5 is synthesized during pregnancy as a stable protein that functions to regulate water secretion during lactation. AQP3 was identified primarily at the basal and lateral membranes of the secretory cells, suggesting a possible involvement in regulated uptake of water and glycerol. AQP1 was identified primarily at the capillary and secretory cell cytoplasmic level and may again be more concerned with uptake and hence milk synthesis, rather than secretion. The fact that expression was developmentally regulated supports, but does not prove, a regulatory involvement of AQPs in water flux through the milk secretory cell.     

Autocrine regulation of mammary cell differentiation

Summary Milk secretion and mammary function in dairy animals are regulated by local mechanisms sensitive to the frequency or efficiency of milking. Acute local control of milk secretion occurs through autocrine feedback inhibition by a milk protein. Sustained changes in milking frequency and milk secretion are associated with longer-term adaptations in the degree of differentiation and, ultimately, the number of mammary epithelial cells. Differentiation of cultured mammary cells is suppressed by a milk fraction containing the inhibitor, suggesting that intra-mammary regulation of differentiation in vivo is elicited by the same autocrine regulator subsequent to its acute effect on milk secretion. The autocrine factor may affect mammary cell differentiation by modulating the number of cell surface hormone receptors for prolactin, thereby changing their sensitivity to circulating hormones.Dedicated to Professor Stuart Patton on the occasion of his 70th birthday.     

The endocrine regulation of milk lipid synthesis and secretion in tammar wallaby (Macropus eugenii)

Lipids in tammar milk are predominantly triacylglycerols, and the fatty acid composition varies during the lactation cycle. Little is known about the regulation of their synthesis. This study investigates the endocrine regulation of lipid synthesis in mammary explants from pregnant tammars. Treatment of mammary explants with insulin resulted in a high level of lipid synthesis, but the lipids accumulated in the cytosol. Culture with prolactin resulted in a small increase in lipid synthesis, but electron microscopy showed lipid globules were synthesized in the mammary epithelial cells and secreted into the lumen. Culture with both insulin and prolactin demonstrated elevated levels of synthesis and secretion of lipid. Analysis of the type of fatty acids synthesized in these mammary explants showed that the initiation of synthesis of C(16:0), which also occurs in the first week of lactation, could be reproduced in the pregnant explants cultured with prolactin alone. However, treatment of mammary explants with hydrocortisone did not show a significant effect on lipid synthesis, secretion or the fatty acid synthesized. These results provide new information identifying the role of insulin and prolactin in regulating milk lipid synthesis and secretion in the tammar.     

Lactation-dependent down regulation of leptin production in mouse mammary gland

Lactation-dependent regulation of leptin expression in mouse mammary gland and parametrial adipose tissue was estimated by RT-PCR analysis for virgin, pregnant, lactating and post-lactating mice, and the serum and milk leptin levels of these mice were also determined by ELISA. Leptin gene expression in mammary gland as well as in adipose tissue was obviously detected before pregnancy, markedly decreased to 30-50% after parturition and kept at the low level during lactation period, and restored to the original level after weaning. The leptin concentration of milk collected just before weaning was about two-fold higher than that of the milk collected at mid-lactating stages. The serum leptin levels of the mid- and late-lactating mice were not significantly higher than those of non-pregnant mice. These results suggested that the lactation-induced down regulation of leptin was associated with autocrine/paracrine action of leptin in mammary and adipose tissues, and that the milk leptin, especially at the latter stages of lactation, was not only ascribed to diffusive transport from maternal blood stream, but also regional production and secretion by mammary epithelial cells. This possible production of leptin by mammary epithelial cells was further supported by the fact that leptin was expressed by cultured cells of mammary epithelial cell line, COMMA-1D, in a manner negatively dependent on the lactogenic hormones.     

Fish oil supplementation of maternal rats on an n-3 fatty acid-deficient diet prevents depletion of maternal brain regional docosahexaenoic acid levels and has a postpartum anxiolytic effect

Docosahexaenoic acid (DHA) and arachidonic acid (AA) are the major polyunsaturated fatty acids (PUFA) in the neuronal membrane. Most DHA and AA accumulation in the brain occurs during the perinatal period via placenta and milk. This study examined whether maternal brain levels of DHA and AA are depleted during pregnancy and lactation due to meeting the high demand of the developing nervous system in the offspring and evaluated the effects of the reproductive cycle on serotonin metabolism and of fish oil (FO) on postpartum anxiety. Pregnant rats were fed during pregnancy and lactation with a sunflower oil-based n-3 PUFA-deficient diet without or with FO supplementation, which provided 0.37% of the energy source as n-3 PUFA, and the age-matched virgin rats were fed the same diets for 41 days. In both sets of postpartum rats, decreased DHA levels compared to those in virgin females were seen in the hypothalamus, hippocampus, frontal cortex, cerebellum, olfactory bulb and retina, while AA depletion was seen only in the hypothalamus, hippocampus and frontal cortex. Serotonin levels were decreased and turnover increased in the brainstem and frontal cortex in postpartum rats compared to virgin rats. FO supplementation during pregnancy and lactation prevented the decrease in maternal brain regional DHA levels, inhibited monoamine oxidase-A activity in the brainstem and decreased anxiety-like behavior. We propose that the reproductive cycle depletes maternal brain DHA levels and modulates maternal brain serotonin metabolism to cause postpartum anxiety and suggest that FO supplementation may be beneficial for postpartum anxiety in women on an n-3 PUFA-deficient diet.