Male-induced early puberty correlates with the maturation of arcuate nucleus kisspeptin neurons in does.

In goats, early exposure of spring-born females to sexually active bucks induces an early puberty onset assessed by the first ovulation. This effect is found when females are continuously exposed well before the male breeding season starting in September. The first aim of this study was to evaluate whether a shortened exposure of females to males could also lead to early puberty. We assessed the onset of puberty in Alpine does isolated from bucks (ISOL), exposed to wethers (CAS), exposed to intact bucks from the end of June (INT1), or mid-August (INT2). Intact bucks became sexually active in mid-September. At the beginning of October, 100% of INT1 and 90% of INT2 exposed does ovulated, in contrast to the ISOL (0%) and CAS (20%) groups. This demonstrated that contact with males that become sexually active is the main factor prompting precocious puberty in females. Furthermore, a reduced male exposure during a short window before the breeding season is sufficient to induce this phenomenon. The second aim was to investigate the neuroendocrine changes induced by male exposure. We found a significant increase in kisspeptin immunoreactivity (fiber density and number of cell bodies) in the caudal part of the arcuate nucleus of INT1 and INT2 exposed females. Thus, our results suggest that sensory stimuli from sexually active bucks (e.g., chemosignals) may trigger an early maturation of the ARC kisspeptin neuronal network leading to gonadotropin-releasing hormone secretion and first ovulation.

Knockdown of regulatory associated protein of TOR (raptor) in hypothalamus-stimulated folliculogenesis and induced ovarian cysts.

CONTEXT: Mammalian target of rapamycin complex 1 (mTORC1) is an essential sensor that regulates fundamental biological processes like cell growth, proliferation and energy metabolism. The treatment of disease by sirolimus, a mTORC1 inhibitor, causes adverse effects, such as female fertility disorders. AIMS: The objective of the study was to decipher the reproductive consequences of a downregulation of mTORC1 in the hypothalamus. METHODS: The reduced expression of mTORC1 was induced after intracerebroventricular injection of lentivirus expressing a short hairpin RNA (shRNA) against regulatory associated protein of TOR (raptor) in adult female mice (ShRaptor mice). KEY RESULTS: The ShRaptor mice were fertile and exhibited a 15% increase in the litter size compared with control mice. The histological analysis showed an increase in antral, preovulatory follicles and ovarian cysts. In the hypothalamus, the GnRH mRNA and FSH levels in ShRaptor mice were significantly elevated. CONCLUSIONS: These results support the hypothesis that mTORC1 in the central nervous system participates in the regulation of female fertility and ovarian function by influencing the GnRH neuronal activity. IMPLICATIONS: These results suggest that a lower mTORC1 activity directly the central nervous system leads to a deregulation in the oestrous cycle and an induction of ovarian cyst development.

Airway Administration of Flagellin Regulates the Inflammatory Response to Pseudomonas aeruginosa.

Excessive lung inflammation and airway epithelial damage are hallmarks of human inflammatory lung diseases, such as cystic fibrosis (CF). Enhancement of innate immunity provides protection against pathogens while reducing lung-damaging inflammation. However, the mechanisms underlying innate immunity-mediated protection in the lung remain mysterious, in part because of the lack of appropriate animal models for these human diseases. TLR5 (Toll-like receptor 5) stimulation by its specific ligand, the bacterial protein flagellin, has been proposed to enhance protection against several respiratory infectious diseases, although other cellular events, such as calcium signaling, may also control the intensity of the innate immune response. Here, we investigated the molecular events prompted by stimulation with flagellin and its role in regulating innate immunity in the lung of the pig, which is anatomically and genetically more similar to humans than rodent models. We found that flagellin treatment modulated NF-κB signaling and intracellular calcium homeostasis in airway epithelial cells. Flagellin pretreatment reduced the NF-κB nuclear translocation and the expression of proinflammatory cytokines to a second flagellin stimulus as well as to Pseudomonas aeruginosa infection. Moreover, in vivo administration of flagellin decreased the severity of P. aeruginosa-induced pneumonia. Then we confirmed these beneficial effects of flagellin in a pathological model of CF by using ex vivo precision-cut lung slices from a CF pigz model. These results provide evidence that flagellin treatment contributes to a better regulation of the inflammatory response in inflammatory lung diseases such as CF.

BDNF and NT3 Reprogram Human Ectomesenchymal Dental Pulp Stem Cells to Neurogenic and Gliogenic Neural Crest Progenitors Cultured in Serum-Free Medium.

BACKGROUND/AIMS: Human Dental Pulp Stem Cells (hDPSCs) are one of the most promising types of cells to regenerate nerve tissues. Standard DMEM+10% fetal bovine serum (FBS) culture medium allows a fast expansion of hDPSC as a surface-adherent cell monolayer. However, the use of FBS also compromises the clinical use of these protocols, and its longterm presence favors hDPSCs differentiation toward mesenchymal cell-derived lineages, at the expense of a reduced capability to generate neural cells. The objective of this work was to characterize the role of neurotrophin signaling on hDPSCs using a serum-free culture protocol, and to assess the neurogenic and gliogenic capacity of hDPSCs for future nerve tissue bioengineering and regeneration. METHODS: We compared the different expression of neurotrophin receptors by RT-PCR, Q-PCR, and IF of hDPSCs cultured with different growth media in the presence or absence of serum. Moreover, we assessed the response of hDPSCs to stimulation of neurotransmitter receptors by live cell calcium imaging under these different media. Finally, we compared the osteogenic potential of hDPSCs by Alizarin red staining, and the differentiation to gliogenic/neurogenic fates by immunostaining for Schwann lineage and neuronal lineage markers. We tested a commercial serum-free medium designed for the growth of mesenchymal stem cells: StemPro MSCTM (STP). RESULTS: hDPSCs cultured in STP generated small non-adherent floating dentospheres that showed very low proliferation rates, in contrast to standard FBS-containing medium. We found that hDPSCs grown in STP conditions overexpressed neurotrophin receptor genes NTRK2 (TrkB) and NTRK3 (TrkC). Interestingly, the stimulation of these receptors by adding their respective ligands BDNF and NT-3 to STP medium enhanced the neural crest (NC) progenitor features of cultured hDPSCs. We observed a 10 to 100-fold increase of migratory NC cell markers HNK1 and P75NTR, and a significant overexpression of pluripotency core factors SOX2, OCT4 and NANOG. Moreover, hDPSCs cultured in BDNF/NT-3 supplemented STP showed a largely increased potential to differentiate towards neuronal and Schwann glial lineage cells, assessed by positive immunostaining for DCX, NeuN and S100ß, p75NTR markers, respectively. CONCLUSION: Our results demonstrate that the use of BDNF and NT-3 combined with STP induced the partial reprogramming of ectomesenchymal hDPSCs to generate early NC progenitor cells, which are far more competent for neuronal and glial differentiation than hDPSCs grown in the presence of FBS.

Pregnancy and estrogen enhance neural progenitor-cell proliferation in the vomeronasal sensory epithelium.

BACKGROUND: The hormonal state during the estrus cycle or pregnancy produces alterations on female olfactory perception that are accompanied by specific maternal behaviors, but it is unclear how sex hormones act on the olfactory system to enable these sensory changes. RESULTS: Herein, we show that the production of neuronal progenitors is stimulated in the vomeronasal organ (VNO) epithelium of female mice during a late phase of pregnancy. Using a wide range of molecular markers that cover the whole VNO cell maturation process in combination with Ca(2+) imaging in early postmitotic neurons, we show that newly generated VNO cells adopt morphological and functional properties of mature sensory neurons. A fraction of these newly generated cells project their axons to the olfactory forebrain, extend dendrites that contact the VNO lumen, and can detect peptides and urinary proteins shown to contain pheromone activity. High-throughput RNA-sequencing reveals concomitant differences in gene expression in the VNO transcriptomes of pregnant females. These include relative increases in expression of 20 vomeronasal receptors, of which 17 belong to the V1R subfamily, and may therefore be considered as candidate receptors for mediating maternal behaviors. We identify the expression of several hormone receptors in the VNO of which estrogen receptor α (Esr1) is directly localized to neural progenitors. Administration of sustained high levels of estrogen, but not progesterone, is sufficient to stimulate vomeronasal progenitor cell proliferation in the VNO epithelium. CONCLUSIONS: Peripheral olfactory neurogenesis driven by estrogen may contribute to modulate sensory perception and adaptive VNO-dependent behaviors during pregnancy and early motherhood.

Cyclic Regulation of Sensory Perception by a Female Hormone Alters Behavior.

Females may display dramatically different behavior depending on their state of ovulation. This is thought to occur through sex-specific hormones acting on behavioral centers in the brain. Whether incoming sensory activity also differs across the ovulation cycle to alter behavior has not been investigated. Here, we show that female mouse vomeronasal sensory neurons (VSNs) are temporarily and specifically rendered "blind" to a subset of male-emitted pheromone ligands during diestrus yet fully detect and respond to the same ligands during estrus. VSN silencing occurs through the action of the female sex-steroid progesterone. Not all VSNs are targeted for silencing; those detecting cat ligands remain continuously active irrespective of the estrous state. We identify the signaling components that account for the capacity of progesterone to target specific subsets of male-pheromone responsive neurons for inactivation. These findings indicate that internal physiology can selectively and directly modulate sensory input to produce state-specific behavior. PAPERCLIP.

Innate Predator Odor Aversion Driven by Parallel Olfactory Subsystems that Converge in the Ventromedial Hypothalamus.

The existence of innate predator aversion evoked by predator-derived chemostimuli called kairomones offers a strong selective advantage for potential prey animals. However, it is unclear how chemically diverse kairomones can elicit similar avoidance behaviors. Using a combination of behavioral analyses and single-cell Ca(2+) imaging in wild-type and gene-targeted mice, we show that innate predator-evoked avoidance is driven by parallel, non-redundant processing of volatile and nonvolatile kairomones through the activation of multiple olfactory subsystems including the Grueneberg ganglion, the vomeronasal organ, and chemosensory neurons within the main olfactory epithelium. Perturbation of chemosensory responses in specific subsystems through disruption of genes encoding key sensory transduction proteins (Cnga3, Gnao1) or by surgical axotomy abolished avoidance behaviors and/or cellular Ca(2+) responses to different predator odors. Stimulation of these different subsystems resulted in the activation of widely distributed target regions in the olfactory bulb, as assessed by c-Fos expression. However, in each case, this c-Fos increase was observed within the same subnuclei of the medial amygdala and ventromedial hypothalamus, regions implicated in fear, anxiety, and defensive behaviors. Thus, the mammalian olfactory system has evolved multiple, parallel mechanisms for kairomone detection that converge in the brain to facilitate a common behavioral response. Our findings provide significant insights into the genetic substrates and circuit logic of predator-driven innate aversion and may serve as a valuable model for studying instinctive fear and human emotional and panic disorders.

Impaired maturation of dendritic spines without disorganization of cortical cell layers in mice lacking NRG1/ErbB signaling in the central nervous system.

Neuregulin-1 (NRG1) and its ErbB2/B4 receptors are encoded by candidate susceptibility genes for schizophrenia, yet the essential functions of NRG1 signaling in the CNS are still unclear. Using CRE/LOX technology, we have inactivated ErbB2/B4-mediated NRG1 signaling specifically in the CNS. In contrast to expectations, cell layers in the cerebral cortex, hippocampus, and cerebellum develop normally in the mutant mice. Instead, loss of ErbB2/B4 impairs dendritic spine maturation and perturbs interactions of postsynaptic scaffold proteins with glutamate receptors. Conversely, increased NRG1 levels promote spine maturation. ErbB2/B4-deficient mice show increased aggression and reduced prepulse inhibition. Treatment with the antipsychotic drug clozapine reverses the behavioral and spine defects. We conclude that ErbB2/B4-mediated NRG1 signaling modulates dendritic spine maturation, and that defects at glutamatergic synapses likely contribute to the behavioral abnormalities in ErbB2/B4-deficient mice.

Requirements for FGF3 and FGF10 during inner ear formation.

Members of the fibroblast growth factor (FGF) gene family control formation of the body plan and organogenesis in vertebrates. FGF3 is expressed in the developing hindbrain and has been shown to be involved in inner ear development of different vertebrate species, including zebrafish, Xenopus, chick and mouse. In the mouse, insertion of a neomycin resistance gene into the Fgf3 gene via homologous recombination results in severe developmental defects during differentiation of the otic vesicle. We have addressed the precise roles of FGF3 and other FGF family members during formation of the murine inner ear using both loss- and gain-of-function experiments. We generated a new mutant allele lacking the entire FGF3-coding region but surprisingly found no evidence for severe defects either during inner ear development or in the mature sensory organ, suggesting the functional involvement of other FGF family members during its formation. Ectopic expression of FGF10 in the developing hindbrain of transgenic mice leads to the formation of ectopic vesicles, expressing some otic marker genes and thus indicating a role for FGF10 during otic vesicle formation. Expression analysis of FGF10 during mouse embryogenesis reveals a highly dynamic pattern of expression in the developing hindbrain, partially overlapping with FGF3 expression and coinciding with formation of the inner ear. However, FGF10 mutant mice have been reported to display only mild defects during inner ear differentiation. We thus created double mutant mice for FGF3 and FGF10, which form severely reduced otic vesicles, suggesting redundant roles of these FGFs, acting in combination as neural signals for otic vesicle formation.

Dampening of cytosolic Ca2+ oscillations on propagation to nucleus.

Ca(2+) signals may regulate gene expression. The increase of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) promotes activation and/or nuclear import of some transcription factors, but others require the increase of the nuclear Ca(2+) concentration ([Ca(2+)](N)) for activation. Whether the nuclear envelope may act as a diffusion barrier for propagation of [Ca(2+)](c) signals remains controversial. We have studied the spreading of Ca(2+) from the cytosol to the nucleus by comparing the cytosolic and the nuclear Ca(2+) signals reported by targeted aequorins in adrenal chromaffin, PC12, and GH(3) pituitary cells. Strong stimulation of either Ca(2+) entry (by depolarization with high K(+) or acethylcholine) or Ca(2+) release from the intracellular Ca(2+) stores (by stimulation with caffeine, UTP, bradykinin, or thyrotropin-releasing hormone (TRH)) produced similar Ca(2+) signals in cytosol and nucleus. In contrast, both spontaneous and TRH-stimulated oscillations of cytosolic Ca(2+) in single GH(3) cells were considerably dampened during propagation to the nucleus. These results are consistent with the existence of a kinetic barrier that filters high frequency physiological [Ca(2+)](c) oscillations without disturbing sustained [Ca(2+)](c) increases. Thus, encoding of the Ca(2+) signal may allow differential control of Ca(2+)-dependent mechanisms located at either the cytosol or the nucleus.