The role of prolactin in the suppression of the response to restraint stress in the lactating mouse.

Suppression of the hypothalamic-pituitary-adrenal (HPA) axis is a well-characterised maternal adaptation that limits the exposure of the offspring to maternally-derived stress hormones. This current study has investigated the possible involvement of the lactogenic hormone, prolactin, in this physiologically important adaptation. As expected, circulating prolactin levels were higher in unstressed lactating mice compared to their virgin counterparts. Interestingly however, the ability of an acute period of restraint stress to further elevate prolactin levels was diminished in the former group. The stress-induced rise in prolactin levels in the virgin animals was concurrent with an increase in prolactin receptor activation within the adrenal cortical cells. This adrenal response was not seen in either the stressed or control lactation group, an observation that may be in part explained by the observed downregulation of prolactin receptor mRNA expression within this tissue. Further evidence of suppression of the HPA axis during lactation was revealed using in situ hybridisation to demonstrate that while acute restraint stress increased corticotrophin releasing hormone (CRH) mRNA expression in the hypothalamic paraventricular nucleus in both virgin and lactating mice, the magnitude of this response was reduced in the latter group. This potentially adaptive response did not, however, appear to result from the altered prolactin profile during lactation because it was not affected by the pharmacological suppression of prolactin secretion from the pituitary. This study therefore suggests that during lactation the response of the HPA axis to stress is suppressed at multiple physiological levels which are mediated by both prolactin-dependent and prolactin-independent mechanisms.

Mating-induced prolactin surge is not required for subsequent neurogenesis in male mice.

Parenting involves major behavioral transitions that are supported by coordinated neuroendocrine and physiological changes to promote the onset of novel offspring-directed behaviors. In comparison to maternal care, however, the mechanisms underlying the transition to paternal care are less understood. Male laboratory mice are predominantly infanticidal as virgins but show paternal responses 2 weeks after mating. Interestingly, males show a mating-induced surge of prolactin, which we hypothesized may be involved in initiating this behavioral transition. During pregnancy, prolactin stimulates olfactory bulb neurogenesis, which is essential for maternal behavior. Mating induces olfactory bulb neurogenesis in males, but it is unknown whether this is driven by prolactin or is important for subsequent paternal care. New olfactory neurons are generated from cells in the subventricular zone (SVZ) and take about 2 weeks to migrate to the olfactory bulb, which may account for the delayed behavioral change in mated males. We investigated whether mating increases cell proliferation at the SVZ. Males were either mated, exposed to receptive female cues, or left alone (control) and injected with Bromodeoxyuridine (BrdU, a marker of cell division). Contrary to our hypothesis, we found that mating decreased cell proliferation in the caudal lateral portion of the SVZ. Next, we tested whether prolactin itself mediates cell proliferation in the SVZ and/or new cell survival in the olfactory bulb by administering bromocriptine (prolactin inhibitor), vehicle, or bromocriptine + prolactin prior to mating. While suppressing prolactin had no effect on cell proliferation in the SVZ, administering exogenous prolactin resulted in significantly higher BrdU-labeled cells in mated but not virgin male mice. No effects of prolactin were observed on new olfactory cell survival. Taken together, prolactin may have context-dependent effects on new cell division in the SVZ, while other unknown mechanisms may be driving the effects on new olfactory cell survival following mating.

Multiple cell types in the oviduct express the prolactin receptor.

Little is known about the physiological role of prolactin in the oviduct. Examining mRNA for all four isoforms of the prolactin receptor (PRLR) in mice by functional oviduct segment and stage of the estrous cycle, we found short form 3 (SF3) to be the most highly expressed, far exceeding the long form (LF) in highly ciliated areas such as the infundibulum, whereas in areas of low ciliation, the SF3 to LF ratio was ~1. SF2 expression was low throughout the oviduct, and SF1 was undetectable. Only in the infundibulum did PRLR ratios change with the estrous cycle. Immunofluorescent localization of SF3 and LF showed an epithelial (both mucosal and mesothelial) distribution aligned with the mRNA results. Despite the high SF3/LF ratio in densely ciliated regions, these regions responded to an acute elevation of prolactin (30 min, intraperitoneal), with LF-tyrosine phosphorylated STAT5 seen within cilia. Collectively, these results show ciliated cells are responsive to prolactin and suggest that prolactin regulates estrous cyclic changes in ciliated cell function in the infundibulum. Changes in gene expression in the infundibulum after prolonged prolactin treatment (7-day) showed prolactin-induced downregulation of genes necessary for cilium development/function, a result supporting localization of PRLRs on ciliated cells, and one further suggesting hyperprolactinemia would negatively impact ciliated cell function and therefore fertility. Flow cytometry, single-cell RNAseq, and analysis of LF-td-Tomato transgenic mice supported expression of PRLRs in at least a proportion of epithelial cells while also hinting at additional roles for prolactin in smooth muscle and other stromal cells.

Elevated prolactin secretion during proestrus in mice: Absence of a defined surge.

Throughout the reproductive cycle in rodents, prolactin levels are generally low. In some species, including rats, a prolactin surge occurs on proestrus with peak concentrations coinciding with the preovulatory luteinizing hormone (LH) surge. In mice, however, there are conflicting reports relating to the occurrence and timing of a proestrous prolactin surge. To gain further insight into the incidence and characteristics of this surge in mice, we have used serial tail tip blood sampling and trunk blood collection from both C57BL/6J (inbred) and Swiss Webster (outbred) mouse strains to build a profile of prolactin secretion during proestrus in individual mice. A clearly defined LH surge was detected in most animals, suggesting the blood sampling approach was suitable for detecting patterns of hormone secretion on proestrus. Despite this, levels of prolactin were quite variable between individuals. Overall both mouse strains showed a generalized rise in prolactin levels on the day of proestrus compared with levels seen in diestrus. This pattern is quite distinct from the discreet, circadian-entrained surge observed in rats.

Patterns of prolactin secretion.

Prolactin is pleotropic in nature affecting multiple tissues throughout the body. As a consequence of the broad range of functions, regulation of anterior pituitary prolactin secretion is complex and atypical as compared to other pituitary hormones. Many studies have provided insight into the complex hypothalamic-pituitary networks controlling prolactin secretion patterns in different species using a range of techniques. Here, we review prolactin secretion in both males and females; and consider the different patterns of prolactin secretion across the reproductive cycle in representative female mammals with short versus long luteal phases and in seasonal breeders. Additionally, we highlight changes in the pattern of secretion during pregnancy and lactation, and discuss the wide range of adaptive functions that prolactin may have in these important physiological states.

Widespread Cell-Specific Prolactin Receptor Expression in Multiple Murine Organs.

The prolactin receptor (Prlr) mediates not only the multiple effects of prolactin, but also those of the placental lactogens and, in humans, some actions of growth hormone. Although Prlr expression has been reported to be widespread in the body, specific cellular expression patterns within tissues are undefined for many organs. One persisting problem in investigating Prlr function is that the protein is difficult to detect using conventional methods. To allow investigation of Prlr expression with a single cell resolution, we have recently developed a knock-in mouse strain in which Cre recombinase is expressed together with the long isoform of the Prlr using an internal ribosome entry site. When crossed to a Cre-dependent reporter mouse strain, Cre-mediated recombination will genetically label cells that acutely express the Prlr as well as cells that have transiently expressed the Prlr during development. We report here the anatomical distribution of cells which express the fluorescent reporter τ green fluorescent protein in a total of 38 organs prepared from young adult male and female Prlr reporter mice. Our results establish a resource for dissecting the functional role of Prlr in multiple murine tissues.

Acute Suppression of LH Secretion by Prolactin in Female Mice Is Mediated by Kisspeptin Neurons in the Arcuate Nucleus.

Hyperprolactinemia causes infertility, but the specific mechanism is unknown. It is clear that elevated prolactin levels suppress pulsatile release of GnRH from the hypothalamus, with a consequent reduction in pulsatile LH secretion from the pituitary. Only a few GnRH neurons express prolactin receptors (Prlrs), however, and thus prolactin must act indirectly in the underlying neural circuitry. Here, we have tested the hypothesis that prolactin-induced inhibition of LH secretion is mediated by kisspeptin neurons, which provide major excitatory inputs to GnRH neurons. To evaluate pulsatile LH secretion, we collected serial blood samples from diestrous mice and measured LH levels by ultrasensitive ELISA. Acute prolactin administration decreased LH pulses in wild-type mice. Kisspeptin neurons in the arcuate nucleus and in the rostral periventricular area of the third ventricle (RP3V) acutely responded to prolactin, but prolactin-induced signaling in kisspeptin neurons was up to fourfold higher in the arcuate nucleus when compared with the RP3V. Consistent with this, conditional knockout of Prlr specifically in arcuate nucleus kisspeptin neurons prevented prolactin-induced suppression of LH secretion. Our data establish that during hyperprolactinemia, suppression of pulsatile LH secretion is mediated by Prlr on arcuate kisspeptin neurons.

Prolactin regulation of insulin-like growth factor 2 gene expression in the adult mouse choroid plexus.

There is clear evidence for carrier-mediated transport of prolactin into the brain, and it has been widely assumed that prolactin receptors (PRLRs) in the choroid plexus (ChP) might mediate this transport. Using PRLR knockout mice, we recently showed that PRLRs in ChP are not required for prolactin transport into the brain. Hence, the function of PRLR in the ChP remains unknown. PRLR expression is increased in the ChP during lactation, suggesting a possible role in adaptive function of prolactin at this time. To gain insight into prolactin function in the ChP, we have utilized RNA sequencing and NanoString techniques to characterize transcriptional changes in response to differing levels of prolactin at diestrus, during pregnancy, and in lactation. We have observed opposing transcriptional effects of prolactin on the ChP in different physiologic states, being primarily inhibitory during diestrus but stimulatory in lactation. Insulin-like growth factor 2 (Igf2), a highly expressing transcript found in the ChP, showed a 6-fold increase at lactation that returned to baseline on suppression of prolactin levels. These results indicate that Igf2 may be an important downstream mediator of prolactin-induced signaling in the ChP.-Phillipps, H. R., Rand, C. J., Brown, R. S. E., Kokay, I. C., Stanton, J.-A., Grattan, D. R. Prolactin regulation of insulin-like growth factor 2 gene expression in the adult mouse choroid plexus.

Analysis of prolactin receptor expression in the murine brain using a novel prolactin receptor reporter mouse.

Prolactin influences a wide range of physiological functions via actions within the central nervous system, as well as in peripheral tissues. A significant limitation in studies investigating these functions is the difficulty in identifying prolactin receptor (Prlr) expression, particularly in the brain. We have developed a novel mouse line using homologous recombination within mouse embryonic stem cells to produce a mouse in which an internal ribosome entry site (IRES) followed by Cre recombinase cDNA is inserted immediately after exon 10 in the Prlr gene, thereby targeting the long isoform of the Prlr. By crossing this Prlr-IRES-Cre mouse with a ROSA26-CAGS-tauGFP (τGFP) reporter mouse line, and using immunohistochemistry to detect τGFP, we were able to generate a detailed map of the distribution of individual Prlr-expressing neurones and fibres throughout the brain of adult mice without the need for amplification of the GFP signal. Because the τGFP is targeted to neurotubules, the labelling detected not only cell bodies, but also processes of prolactin-sensitive neurones. In both males and females, Cre-dependent τGFP expression was localised, with varying degrees of abundance, in a number of brain regions, including the lateral septal nucleus, bed nucleus of the stria terminalis, preoptic and hypothalamic nuclei, medial habenula, posterodorsal medial amygdala, and brainstem regions such as the periaqueductal grey and parabrachial nucleus. The labelling was highly specific, occurring only in cells where we could also detect PrlrmRNA by in situ hybridisation. Apart from two brain areas, the anteroventral periventricular nucleus and the medial preoptic nucleus, the number and distribution of τGFP-immunopositive cells was similar in males and females, suggesting that prolactin may have many equivalent functions in both sexes. These mice provide a valuable tool for investigating the neural circuits underlying the actions of prolactin.

Prolactin action in the medial preoptic area is necessary for postpartum maternal nursing behavior.

Pregnancy hormones, such as prolactin, sensitize neural circuits controlling parental interactions to induce timely activation of maternal behaviors immediately after parturition. While the medial preoptic area (MPOA) is known to be critical for maternal behavior, the specific role of prolactin in this brain region has remained elusive. Here, we evaluated the role of prolactin action in the MPOA using complementary genetic strategies in mice. We characterized prolactin-responsive neurons within the MPOA at different hormonal stages and delineated their projections in the brain. We found that MPOA neurons expressing prolactin receptors (Prlr) form the nexus of a complex prolactin-responsive neural circuit, indicating that changing prolactin levels can act at multiple sites and thus, impinge on the overall activity of a distributed network of neurons. Conditional KO of Prlr from neuronal subpopulations expressing the neurotransmitters GABA or glutamate within this circuit markedly reduced the capacity for prolactin action both in the MPOA and throughout the network. Each of these manipulations, however, produced only subtle impacts on maternal care, suggesting that this distributed circuit is robust with respect to alterations in prolactin signaling. In contrast, acute deletion of Prlr in all MPOA neurons of adult female mice resulted in profound deficits in maternal care soon after birth. All mothers abandoned their pups, showing that prolactin action on MPOA neurons is necessary for the normal expression of postpartum maternal behavior in mice. Our data establish a critical role for prolactin-induced behavioral responses in the maternal brain, ensuring survival of mammalian offspring.

Conditional Deletion of the Prolactin Receptor Reveals Functional Subpopulations of Dopamine Neurons in the Arcuate Nucleus of the Hypothalamus.

UNLABELLED: Tuberoinfundibular dopamine (TIDA) neurons, known as neuroendocrine regulators of prolactin secretion from the pituitary gland, also release GABA within the hypothalamic arcuate nucleus. As these neurons express prolactin receptors (Prlr), prolactin may regulate GABA secretion from TIDA neurons, potentially mediating actions of prolactin on hypothalamic function. To investigate whether GABA is involved in feedback regulation of TIDA neurons, we examined the physiological consequences of conditional deletion of Prlr in GABAergic neurons. For comparison, we also examined mice in which Prlr were deleted from most forebrain neurons. Both neuron-specific and GABA-specific recombination of the Prlr gene occurred throughout the hypothalamus and in some extrahypothalamic regions, consistent with the known distribution of Prlr expression, indicative of knock-out of Prlr. This was confirmed by a significant loss of prolactin-induced phosphorylation of STAT5, a marker of prolactin action. Several populations of GABAergic neurons that were not previously known to be prolactin-sensitive, notably in the medial amygdala, were identified. Approximately 50% of dopamine neurons within the arcuate nucleus were labeled with a GABA-specific reporter, but Prlr deletion from these dopamine/GABA neurons had no effect on feedback regulation of prolactin secretion. In contrast, Prlr deletion from all dopamine neurons resulted in profound hyperprolactinemia. The absence of coexpression of tyrosine hydroxylase, a marker for dopamine production, in GABAergic nerve terminals in the median eminence suggested that rather than a functional redundancy within the TIDA population, the dopamine/GABA neurons in the arcuate nucleus represent a subpopulation with a functional role distinct from the regulation of prolactin secretion. SIGNIFICANCE STATEMENT: Using a novel conditional deletion of the prolactin receptor, we have identified functional subpopulations in hypothalamic dopamine neurons. Although commonly considered a uniform population of neuroendocrine neurons involved in the control of prolactin secretion, we have shown that approximately half of these neurons express GABA as well as dopamine, but these neurons are not necessary for the feedback regulation of prolactin secretion. The absence of tyrosine hydroxylase in GABAergic nerve terminals in the median eminence suggests that only the non-GABAergic dopamine neurons are involved in the control of pituitary prolactin secretion, and the GABAergic subpopulation may function as interneurons within the arcuate nucleus to regulate other aspects of hypothalamic function.

Maintained expression of genes associated with metabolism in the ventromedial hypothalamic nucleus despite development of leptin resistance during pregnancy in the rat.

Hyperphagia and weight gain to acquire energy stores for development and growth of the fetus and to prepare for the demands of lactation are important adaptations to support a healthy pregnancy. As a consequence, hypothalamic leptin resistance develops to enable maintenance of a positive energy state. During pregnancy there is a decrease in leptin receptor expression and reduced leptin-induced phospho signal transducer and activator of transcription 3 (pSTAT3) in the ventromedial nucleus of the hypothalamus (VMN), suggesting that the VMN is a key site of pregnancy-induced modification in the control of energy homeostasis. The aim of this study was to investigate expression levels of known gene targets, which are involved in metabolic regulation and glucosensing, within the VMN during pregnancy. Using in situ hybridization, pituitary adenylate cyclase-activated polypeptide (Pacap), brain-derived neurotrophic factor (Bdnf), and glucokinase messenger ribonucleic acid (mRNA) expression were localized in the hypothalamus of nonpregnant and day 14 pregnant rats, then expression levels were compared by quantitative polymerase chain reaction (qPCR) using laser capture microdissection of the VMN and arcuate nucleus. Despite significantly elevated plasma leptin and insulin concentrations, and lower blood glucose levels, during pregnancy, no significant changes in gene expression of Pacap, Bdnf, or glucokinase were detected between nonpregnant and day 14 pregnant groups. These data suggest that loss of leptin and insulin sensitivity in the VMN might allow gene expression to be maintained at normal/control levels in this nucleus, despite marked changes in the levels of these important regulatory hormones. These data provide further evidence for development of leptin resistance in the VMN as an adaptive response during pregnancy.

XIAP: a potential determinant of ovarian follicular fate.

X-linked inhibitor of apoptosis protein (XIAP), a member of the inhibitor of apoptosis protein family, is involved in regulating a number of functions including receptor-mediated intracellular signalling and early development. Its role as an endogenous caspase inhibitor, however, is the most highly characterised. Consequently, this protein has been implicated as an anti-apoptotic factor in the ovary. In vitro and in vivo studies have begun dissecting the stimuli and signalling networks that lead to XIAP upregulation in granulosa cells. The objective of this review is to briefly summarise the current knowledge concerning XIAP and its interactions with different caspases. Furthermore, XIAP's emerging role in the mammalian ovary will be explored and comparison is made with its functions in the mammary gland. Finally, the idea that XIAP may act as a molecular signalling switch in granulosa cells following detachment from underlying layers to promote follicular atresia will be introduced.

X-linked inhibitor of apoptosis protein and active caspase-3 expression patterns in antral follicles in the sheep ovary.

X-linked inhibitor of apoptosis protein (XIAP) interacts with caspases to inhibit their activity, thereby providing a potential mechanism for regulation of granulosa cell apoptosis occurring during follicular atresia. The aim of this study was to determine the presence and localization of XIAP mRNA and protein content in the sheep ovary and compare these expression patterns with active caspase-3 protein in the same antral follicles. Romney ewe estrous cycles (n=25) were synchronized with 2-3 Estrumate injections and ovarian tissue collected during the luteal and follicular phases of the cycle. The presence of XIAP mRNA was confirmed by RT-PCR using laser capture microdissected ovarian cell samples. XIAP mRNA was subsequently localized by in situ hybridization histochemistry and XIAP and active caspase-3 protein visualized by immunohistochemistry. In antral follicles extensive XIAP localization was evident in both granulosa and thecal cells. In contrast, mRNA expression was widespread in granulosa cells and only detected in thecal tissue from a small proportion of antral follicles. Active caspase-3 and XIAP comparative expression analysis showed positive XIAP mRNA expression in all late luteal phase (day 14) follicles, despite varying levels of active caspase-3 protein. A proportion of follicular phase (days 15 and 16) follicles, however, showed an inverse expression relationship at the protein and mRNA levels in both granulosa and thecal tissue, as did XIAP protein in day 14 follicles. These results suggest high XIAP may prevent activation of caspase-3, thereby regulating follicular atresia in antral follicles and could potentially be utilized as a marker of follicular health.