Mothers and offspring: The rabbit as a model system in the study of mammalian maternal behavior and sibling interactions

This article is part of a Special Issue “Parental Care”.Jay Rosenblatt effectively promoted research on rabbit maternal behavior through his interaction with colleagues in Mexico. Here we review the activities of pregnant and lactating rabbits (Oryctolagus cuniculus), their neuro-hormonal regulation, and the synchronization of behavior between mother and kits. Changing concentrations of estradiol, progesterone, and prolactin throughout gestation regulate nest-building (digging, straw-carrying, fur-pulling) and prime the mother's brain to respond to the newborn. Nursing is the only mother–young contact throughout lactation. It happens once/day, inside the nest, with ca. 24 h periodicity, and lasts around 3 min. Periodicity and duration of nursing depend on a threshold of suckling as procedures reducing the amount of nipple stimulation interfere with the temporal aspects of nursing, though not with the doe's maternal motivation. Synchronization between mother and kits, critical for nursing, relies on: a) the production of pheromonal cues which guide the young to the mother's nipples for suckling; b) an endogenous circadian rhythm of anticipatory activity in the young, present since birth. Milk intake entrains the kits' locomotor behavior, corticosterone secretion, and the activity of several brain structures. Sibling interactions within the huddle, largely determined by body mass at birth, are important for: a) maintaining body temperature; b) ensuring normal neuromotor and social development. Suckling maintains nursing behavior past the period of abundant milk production but abrupt and efficient weaning occurs in concurrently pregnant-lactating does by unknown factors. Conclusion: female rabbits have evolved a reproductive strategy largely dissociating maternal care from maternal presence, whose multifactorial regulation warrants future investigations.     

Maternal care of rabbits in the lab and on the farm: endocrine regulation of behavior and productivity

Maternal behavior in rabbits has been well described in the wild, the laboratory, and the farm. Salient characteristics include: (a) the construction of a nest (inside an underground burrow or a box), composed of straw/grass and body hair and (b) the display of a single, brief (ca. 3 min) nursing bout per day. The onset and decline of nest-building in mid and late pregnancy are controlled by specific combinations of estradiol, progesterone, testosterone, and prolactin. Following parturition (kindling) does can mate and become pregnant again. Management strategies have been used on the farm to enhance productivity, impinging on specific reproductive processes (e.g., use of hormones to synchronize estrus, artificial insemination at kindling, doe-litter separation on specific days of lactation to increase sexual receptivity and fertility). Knowledge about the rabbit's reproductive physiology and behavior will be enriched by integrating research coming from the laboratory (where estrus, pregnant-only, or lactating-only animals are the main categories investigated) with studies performed on the farm or research station (where pregnant-lactating rabbits are prevalent). Similarly, the high productivity demands of modern rabbit farms will benefit from the information obtained in the laboratory where specific issues in reproductive neuroendocrinology are explored with methodologies that are not amenable for farm use.     

Androgens stimulate specific aspects of maternal nest-building and reduce food intake in rabbits

Fluctuations in the plasma concentration of estradiol, progesterone, and prolactin across pregnancy regulate maternal nest-building (digging, straw-carrying, and hair-plucking) and food intake in rabbits. Because testosterone levels also change through pregnancy, we investigated if the injection of testosterone propionate (TP; 1 or 5 mg/day) or 5alpha-dihydrotestosterone propionate (5 or 10 mg/day) for 20 days, alone and combined with progesterone (P; 10 mg/day from days 2 to 15), modulated nest-building and food intake in ovariectomized rabbits. Only the combined injection of TP (5 mg/day) plus P stimulated digging and no treatment promoted straw-carrying or hair-plucking. Both androgens induced hair-loosening from the ventrum, an effect counteracted by P. High doses of TP and 5alpha-dihydrotestosterone propionate reduced food intake by 60-70% of baseline values; this effect was counteracted by P in TP-treated animals. These results support a participation of androgens in specific aspects of maternal nest-building and reveal a strong inhibitory effect on food intake.     

Prolactin receptors in the brain during pregnancy and lactation: implications for behavior

Numerous studies have documented prolactin regulation of a variety of brain functions, including maternal behavior, regulation of oxytocin neurons, regulation of feeding and appetite, suppression of ACTH secretion in response to stress, and suppression of fertility. We have observed marked changes in expression of prolactin receptors in specific hypothalamic nuclei during pregnancy and lactation. This has important implications for neuronal functions regulated by prolactin. In light of the high circulating levels of prolactin during pregnancy and lactation and the increased expression of prolactin receptors in the hypothalamus, many of these functions may be enhanced or exaggerated in the maternal brain. The adaptations of the maternal brain allow the female to exhibit the appropriate behavior to feed and nurture her offspring, to adjust to the nutritional and metabolic demands of milk production, and to maintain appropriate hormone secretion to allow milk synthesis, secretion, and ejection. This review aims to summarize the evidence that prolactin plays a key role in regulating hypothalamic function during lactation and to discuss the hypothesis that the overall role of prolactin is to organize and coordinate this wide range of behavioral and neuroendocrine adaptations during pregnancy and lactation.     

Elevated Fos-like immunoreactivity in the brains of postpartum female prairie voles, Microtus ochrogaster

This study investigated the expression of the protein product of the immediate early gene c-fos in the brains of female prairie voles (Microtus ochrogaster) in association with pregnancy and postparturient activities including maternal behavior, lactation and postpartum estrus. Fos expression was assessed in female voles that were late in pregnancy, nonpregnant or at one of three different times postpartum (0-8, 12-24, and 24-48 h, respectively). A significant increase in the number of cells displaying Fos immunoreactivity (Fos-ir) was observed during the 0-8 h and 12-24 h postpartum time periods in the accessory olfactory bulbs, medial preoptic area, hypothalamus (specifically, the supraoptic nucleus, ventro-medial hypothalamus, and paraventricular nucleus), lateral septum, bed nucleus of the stria terminalis, and primary somatosensory area of the brain. The number of Fos-ir cells decreased after 24 h postpartum. There were no significant changes in Fos-ir cell numbers in the primary olfactory bulbs, hippocampus, or caudate putamen. The neural activation of the medial preoptic area, accessory olfactory bulbs, hypothalamus, and bed nucleus is consistent with reports in rats of Fos induction associated with the onset of maternal behavior. In voles postpartum estrous behavior begins and ends 0-12 h after parturition. Maternal behavior, including lactation, is initiated at the same time but persists for several weeks. The highest Fos-ir cell numbers reported here coincide with the timing of postpartum estrous behavior in this species.     

Hormone profiles and nest-building behavior during the periparturient period in rabbit does

This study was done to elucidate the relationship between the hormones beta-estradiol, progesterone, prolactin and beta-endorphin and nest-building behavior in rabbit does during the periparturient period. Beta-estradiol increased as parturition approached with a significant increase starting 2 days before parturition (day -2). Progesterone decreased with the progress of gestation, but a significant decrease was observed starting on day -2. Prolactin concentration started to increase on day -2 prepartum but a significant increase in prolactin concentration was not noted until the last day of pregnancy. The concentration of beta-endorphin was highly variable during the sampling period and could not be correlated with the other hormones or nest-building behavior. To assess the role of prolactin in nest-building behavior, groups of rabbits were treated with bromocryptine, bromocryptine plus prolactin or saline (controls). Treatment with 4 mg bromocryptine or 4 mg bromocryptine + 1.5 mg bovine prolactin on days 25 and 27 of pregnancy did not affect the number of live kits born or the gestation length. The mean nest scores, a measure of the nest quality, were not affected by bromocryptine treatment, but treatment with bromocryptine plus prolactin resulted in lower nest scores (P < 0.05). Injection of 8 mg bromocryptine from day 28 of gestation to kindling resulted in an extended gestation (P < 0.05). Injection of 4 or 8 mg bromocryptine resulted in fewer live kits born (P < 0.05), reduced nest scores (P < 0.01) and blocked milk production, as determined from the palpable mammary tissue. These results indicate that prolactin has less influence on nest-building behavior than on milk production. The hormones most likely to influence nest building are beta-estradiol and progesterone because the levels of these hormones started to change at the time when the rabbits started to prepare nests. Further study is required to determine the influence of these hormones on nest-building behavior.     

Maternal Responsiveness Increases during Pregnancy and after Estrogen Treatment in Macaques

Maternal responsiveness in primates has long been considered emancipated from endocrine factors and entirely dependent on experience and cognition. Here we report that group-living pigtail macaque females increased their rate of interaction with infants in the last weeks of pregnancy in correspondence with an increase in plasma levels of estradiol and progesterone. Estrogen treatment increased the rate at which ovariectomized rhesus females interacted with infants. This is the first evidence that steroid hormones influence maternal responsiveness in anthropoid primates. All untreated ovariectomized females and nonpregnant females interacted with infants, indicating that although estrogen can enhance responsiveness to infants, ovarian or pregnancy hormones are not necessary for the expression of infant-directed behavior in female macaques. The findings of this study suggest fundamental similarities, rather than differences, in the endocrine modulation of maternal responsiveness in primates and other mammals.     

Melanin-concentrating hormone in the medial preoptic area reduces active components of maternal behavior in rats

Melanin-concentrating hormone (MCH) is an inhibitory neuropeptide mainly synthesized in neurons of the lateral hypothalamus and incerto-hypothalamic area of mammals that has been implicated in behavioral functions related to motivation. During lactation, this neuropeptide is also expressed in the medial preoptic area (mPOA), a key region of the maternal behavior circuitry. Notably, whereas MCH expression in the mPOA progressively increases during lactation, maternal behavior naturally declines, suggesting that elevated MCHergic activity in the mPOA inhibit maternal behavior in the late postpartum period. To explore this idea, we assessed the maternal behavior of early postpartum females following bilateral microinfusions of either MCH (50 and 100 ng/0.2 μl/side) or the same volume of vehicle into the mPOA. As expected, females receiving 100 ng MCH into the mPOA exhibited significant deficits in the active components of maternal behavior, including retrieving and nest building. In contrast, nursing, as well as other behaviors, including locomotor activity, exploration, and anxiety-like behavior, were not affected by intra-mPOA MCH infusion. The present results, together with previous findings showing elevated expression of this neuropeptide toward the end of the postpartum period, suggest that modulation of mPOA function by MCH may contribute to the weaning of maternal responsiveness characteristic of the late postpartum period.     

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.     

Maternal olfaction differentially modulates oxytocin and prolactin release during suckling in goats

In postparturient goats, olfactory recognition of the young allows the establishment of a selective bond between the mother and her kids. Once this bond is formed, the mother rejects alien young that attempt to suckle. We tested whether the development of the maternal selective bond in goats modulates prolactin (PRL) and oxytocin (OT) release in response to suckling. On day 37 of lactation, serial blood samples were taken during nursing of the mother's own or alien kid(s) in 10 intact/selective goats and in 10 goats rendered anosmic/nonselective through prepartum peripheral ZnSO(4) irrigation. Spontaneous nursing behavior was also studied weekly from day 7 to 30 of lactation, at which time milk production was measured. Maternal selectivity had no effect on PRL release, in contrast to OT release, which was significantly affected by this factor. Intact mothers released OT only when nursing their own kids, but not with aliens, while anosmic/nonselective dams showed an increase in OT levels regardless of the identity of the kids. In addition to these effects on maternal selectivity, the amplitude of the response of both hormones was lower in anosmic mothers than in intact mothers. Finally, nursing behavior and milk production were not significantly affected by anosmia. We conclude that maternal selective behavior in goats, which relies on the individual olfactory signature of the kid, modulates the OT, but not the PRL, response to suckling. In addition, perception of the smell of the young appears to have a general facilitatory effect, independent of the kid's identity, on the release of both hormones.     

Estrogen implants in the lateral habenular nucleus do not stimulate the onset of maternal behavior in female rats

The natural onset of maternal behavior in the rat is hormonally mediated. Estrogen, progesterone, and prolactin administered to ovariectomized females in amounts and sequences that produce circulating levels similar to those found during pregnancy stimulate the onset of maternal behavior. In fact, maternal behavior can be stimulated by estrogen alone, administered either peripherally or by implant in the central nervous system. The lateral habenula (Lhb), which is a necessary component in the neural circuit that supports maternal behavior, contains a subset of neurons with estrogen receptors. The present study investigated whether estradiol implants directly in the Lhb are sufficient to stimulate maternal behavior. Female rats, hysterectomized and ovariectomized on day 16 of pregnancy, received estrogen implants in the Lhb or, as a positive control, in the medial preoptic area (MPOA). An additional control group received cholesterol implants in the Lhb. All females were tested for pup retrieval, nest building, crouching behavior, locomotor activity, and carrying behavior. Estradiol implants into the Lhb did not stimulate the onset of maternal behavior. Females with estrogen implants in the Lhb scored significantly lower in pup retrieval and crouching behavior compared to females with implants in the MPOA and were not significantly different from females with cholesterol implants in the Lhb. There were also no significant differences in overall activity or carrying behavior among the groups.     

Olfactory regulation of maternal behavior in mammals

In mammals, olfactory cues are extensively used in many aspects of maternal care to ensure the coordination of mother-infant interactions and consequently the normal development of the offspring. Outside the period of parturition and lactation, when the young are not a behavioral priority, olfactory cues play an inhibitory role on maternal responsiveness since in most mammalian species studied so far, nonpregnant females find the odor of young aversive. On the contrary at the time of parturition, a shift in the hedonic value of infantile odors occurs so that the young now become a very potent stimulus and this sensorial processing constitutes an important part of the maternal motivational system. Moreover, infants' odors provide a basis for individual recognition by their mothers and some species (ungulates) have developed highly specialized mechanisms for processing of the infant signals. Perception of the smell of the young also regulates various aspects of maternal behavior. Dodecyl propionate, a compound released by of pup's preputial glands, has been shown to influence anogenital licking behavior, a fundamental pattern of maternal behavior in rodents. While there is no functional specificity of either the main or the accessory olfactory systems in the development of maternal behavior amongst species, it appears that only the main olfactory system is implicated when individual odor discrimination of the young is required. Neural structures, such as the main olfactory bulb, undergo profound changes when exposed to offspring odors at parturition. These changes in synaptic circuitry contribute both to maternal responsiveness to these odors, to their memorization, and to effects of long-term maternal experience.     

Cytosol and nuclear estrogen receptor binding in the preoptic area and hypothalamus of female rats during pregnancy and ovariectomized, nulliparous rats after steroid priming: correlation with maternal behavior

In a previous study, high nuclear estrogen receptor concentrations in the preoptic area (POA) were found on Day 16 of pregnancy to prime females to respond to a subsequent low dose of estradiol benzoate (EB) after hysterectomy-ovariectomy by exhibiting maternal behavior in 48 hr. Receptor concentrations in the POA were found to be higher than those in the hypothalamus (HYP). The present study investigated when nuclear estrogen receptors increase during pregnancy in POA and when the difference in receptor concentrations between POA and HYP occurs. An attempt was made to reproduce these pregnancy changes with a 16-day treatment of estrogen and progesterone in ovariectomized (OVX), nulliparous rats. In Experiment 1, we measured cytosol and nuclear estrogen receptor concentrations in the POA and HYP of female rats during pregnancy. Nuclear receptor concentrations in the POA increased beginning on Day 10, increased again on Day 16, and continued at this high level for the remainder of pregnancy. Nuclear estrogen receptor concentrations in the HYP remained at a lower level throughout most of pregnancy until Day 22 when they increased significantly. In Experiment 2, we tested the maternal behavior and measured estrogen receptor concentrations in OVX, steroid-primed, nulliparous rats after hysterectomy (H) and EB treatment. While 90% of estradiol (E) + progesterone (P)-primed females displayed short-latency maternal behavior 48 hr after H and EB treatment, 46% of E + vehicle (V)-treated controls were maternal. At 0 hr (prior to H and EB treatment), there was a significantly larger nuclear receptor accumulation in the POA but significantly attenuated receptor binding in the HYP. P treatment significantly affected cytosol and nuclear estrogen receptor dynamics. Differences in nuclear estrogen receptor concentrations were shown to be based on the number of available binding sites and not to changes in receptor affinity for estradiol.     

Diminished maternal responsiveness during pregnancy in multiparous female common marmosets

Common marmoset monkeys (Callithrix jacchus) live in small groups in which, usually, only a single female breeds and all group members provide infant care. When two females breed concurrently, however, they may commonly kill one another's infants, especially during the peripartum period. To investigate the mechanisms underlying infanticide by breeding females, we characterized responses of multiparous females to infants and determined circulating hormone levels in adult females during early pregnancy, late pregnancy, and the early postpartum period. Additionally, we compared the responses of postpartum females to their own infants and infants of other females (unfamiliar infants). Postpartum females were highly maternal toward both their own and unfamiliar infants, and showed no differences in their behavioral or hormonal responses to the two. During both early and late pregnancy, however, these females exhibited longer latencies to initially approach unfamiliar infants and spent less time carrying unfamiliar infants. Moreover, females spent less time carrying unfamiliar infants during late pregnancy than early pregnancy. Most late pregnant females never carried infants, and those that did rejected them quickly. Prolactin concentrations were higher and progesterone concentrations lower postpartum than in early or late pregnancy, while estradiol concentrations, the estradiol-to-progesterone ratio, and cortisol levels were higher during late pregnancy. Within reproductive conditions, however, maternal behaviors were not correlated with hormone levels. These results suggest that maternal responsiveness in marmosets may be attenuated during pregnancy, especially late pregnancy, and this may contribute to infanticide by breeding females.     

Progesterone Receptor Expression Declines in the Guinea Pig Uterus during Functional Progesterone Withdrawal and in Response to Prostaglandins

Progesterone withdrawal is essential for parturition, but the mechanism of this pivotal hormonal change is unclear in women and other mammals that give birth without a pre-labor drop in maternal progesterone levels. One possibility suggested by uterine tissue analyses and cell culture models is that progesterone receptor levels change at term decreasing the progesterone responsiveness of the myometrium, which causes progesterone withdrawal at the functional level and results in estrogen dominance enhancing uterine contractility. In this investigation we have explored whether receptor mediated functional progesterone withdrawal occurs during late pregnancy and labor in vivo. We have also determined whether prostaglandins that induce labor cause functional progesterone withdrawal by altering myometrial progesterone receptor expression. Pregnant guinea pigs were used, since this animal loses progesterone responsiveness at term and gives birth in the presence of high maternal progesterone level similarly to primates. We found that progesterone receptor mRNA and protein A and B expression decreased in the guinea pig uterus during the last third of gestation and in labor. Prostaglandin administration reduced while prostaglandin synthesis inhibitor treatment increased progesterone receptor A protein abundance. Estrogen receptor-1 protein levels remained unchanged during late gestation, in labor and after prostaglandin or prostaglandin synthesis inhibitor administration. Steroid receptor levels were higher in the non-pregnant than in the pregnant uterine horns. We conclude that the decreasing expression of both progesterone receptors A and B is a physiological mechanism of functional progesterone withdrawal in the guinea pig during late pregnancy and in labor. Further, prostaglandins administered exogenously or produced endogenously stimulate labor in part by suppressing uterine progesterone receptor A expression, which may cause functional progesterone withdrawal, promote estrogen dominance and foster myometrial contractions.     

Prolactin receptor expression in monolayer cultures of rabbit mammary epithelial cells: pre- and postpartum [125I]-prolactin binding activity

Expression of specific [125I]-prolactin-binding sites under culture conditions has been investigated for isolated mammary epithelial cells from virgin, pregnant, and lactating rabbits. Primary monolayer cultures were obtained by sequential enzymatic dispersion of mammary tissue followed by 48 hr incubation in a medium selective for epithelial cells. Scatchard analyses of binding data obtained from these cultures indicated a single class of receptor sites, the affinity constant of which (2.5 X 10(9) M-1) did not vary significantly during mammary development. The number of prolactin receptors, however, expressed by virgin and early pregnant epithelial cells was significantly increased over those from late pregnancy or lactation. Less differentiated cells also respond to growth in pregnant rabbit serum with an increase in specific [125I]-prolactin binding. The diminished receptor expression by cells obtained after 17 days of pregnancy coincides with the attainment of secretory capacity in the animal, and may reflect the influence of the low serum prolactin or high progesterone levels circulating during the last trimester in the rabbit, or be the cultural expression of secretory differentiation.     

Experience-facilitated improvements in pup retrieval; evidence for an epigenetic effect

The quality and quantity of maternal care received during infancy are highly predictive of successful infant development. It has been well established, primarily in rats, that the combination of hormonal and infant stimuli at birth modifies neural circuits that regulate maternal responsiveness. During subsequent interactions, infant stimuli are more likely to elicit rapid maternal responsiveness. Some species, such as humans, can display maternal care in the absence of the endocrine events of pregnancy and birth. Similarly, virgin C57BL/6J female mice, display maternal care toward infants, and experience with infants elicits long-lasting increases in maternal care. We hypothesized that these experience-induced changes in behavior may be mediated by chromatin modifications, which in turn change expression of genes that promote maternal care. One site of action is the medial preoptic area (MPOA). To test our hypothesis we treated virgin female mice with sodium butyrate, a histone deacetylase inhibitor. This treatment potentiated maternal responsiveness as well as the expression of several genes: estrogen receptor β (Esr2), oxytocin (Oxt), and cyclicAMP response element binding protein (CREB) binding protein (Crebbp; a histone acetyltransferase) in the MPOA. These data suggest that experience induces high levels of maternal care via epigenetic modifications.     

Plasma prolactin concentrations and cyclic activity in pony mares during parturition and early lactation

Five pony mares were blood sampled during late pregnancy, foaling and early lactation. An homologous assay for horse prolactin was used to measure plasma prolactin concentrations in these samples. Regular estimates of cyclic activity were also made. Plasma prolactin concentrations rose markedly in the last week of pregnancy and remained high although variable in early lactation, before declining to basal levels by 1-2 months post partum. All mares showed a post-partum oestrus 7.0 +/- 0.9 days after parturition. One mare whose foal died shortly after birth showed a rapid decline in plasma prolactin values after death of the foal and an early oestrous period (4 days after parturition). The pattern of prolactin changes reported for the mare are in agreement with those reported for other mammalian species.