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Müllerian ducts are paired tubular structures that give rise to most of the female reproductive organs. Any abnormalities in the development and differentiation of these ducts lead to anatomical defects in the female reproductive tract organs categorized as Müllerian duct anomalies. Due to the limited access to fetal tissues, little is understood of human reproductive tract development and the associated anomalies. Although organoids represent a powerful model to decipher human development and disease, such organoids from fetal reproductive organs are not available. Here, we developed organoids from human fetal fallopian tubes and uteri and compared them with their adult counterparts. Our results demonstrate that human fetal reproductive tract epithelia do not express some of the typical markers of adult reproductive tract epithelia. Furthermore, fetal organoids are grossly, histologically, and proteomically different from adult organoids. While external supplementation of WNT ligands or activators in culture medium is an absolute requirement for the adult reproductive tract organoids, fetal organoids are able to grow in WNT-deficient conditions. We also developed decellularized tissue scaffolds from adult human fallopian tubes and uteri. Transplantation of fetal organoids onto these scaffolds led to the regeneration of the adult fallopian tube and uterine epithelia. Importantly, suppression of Wnt signaling, which is altered in patients with Müllerian duct anomalies, inhibits the regenerative ability of human fetal organoids and causes severe anatomical defects in the mouse reproductive tract. Thus, our fetal organoids represent an important platform to study the underlying basis of human female reproductive tract development and diseases.
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Trompas Uterinas , Conductos Paramesonéfricos , Organoides , Útero , Adulto , Animales , Trompas Uterinas/crecimiento & desarrollo , Femenino , Feto , Humanos , Ligandos , Ratones , Conductos Paramesonéfricos/anomalías , Organoides/crecimiento & desarrollo , Organoides/metabolismo , Útero/crecimiento & desarrollo , Vía de Señalización WntRESUMEN
In Brief: In many mammals, the lipid platelet-activating factor (PAF) has important functions in female reproduction and fertility. This study shows that PAF is present in the reproductive tissues of mares and is involved in processes related to ovulation and early pregnancy. Abstract: Platelet-activating factor (PAF) has been implicated in a number of reproductive processes ranging from ovulation to embryo motility but has not been widely explored in the mare. To identify the presence and examine the role of PAF in the equine periconception processes, targeted mass spectrometry coupled with chromatographic separation was performed on equine follicular fluid (FF), and PAF was quantitatively detected. Subsequently, untargeted high-resolution mass spectrometry-based lipidomic analysis was carried out to quantify PAF in different-sized pre-ovulatory follicles, whereby different molecular species of PAF, PAF (14:0) and PAF (16:1), were both seen to be increasing with follicle diameter. These findings suggest that PAF within FF is increasing as preovulatory follicles approach ovulation. Additionally, immunofluorescence staining identified the PAF receptor in the luminal pericellular, apical, and basal aspect of equine oviductal epithelial cells. Lastly, an equine oviductal epithelial organoid model was generated and showed that the addition of PAF significantly increased the ciliary beat frequency (CBF) (Hz), an action consistent with a role for PAF in embryo migration. It is proposed that the local action of PAF on the ciliated cells of the oviduct propels both the oocyte and the conceptus towards the uterus. In the mare, it appears that PAF is a contributor during the periconception period, potentially being a mediator in the mechanisms of ovulation and in the dialogue of very early pregnancy.
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Ovulación , Factor de Activación Plaquetaria , Animales , Caballos/fisiología , Femenino , Factor de Activación Plaquetaria/metabolismo , Factor de Activación Plaquetaria/farmacología , Embarazo , Ovulación/fisiología , Folículo Ovárico/metabolismo , Folículo Ovárico/fisiología , Líquido Folicular/metabolismo , Fertilización/fisiologíaRESUMEN
Organoid technology has provided a unique opportunity to study early human development and decipher various steps involved in the pathogenesis of disease. The technology is already used in clinics to improve human patient outcomes. However, limited knowledge of the methodologies required to establish organoid culture systems in domestic animals has slowed the advancement and application of organoid technology in veterinary medicine. This is particularly true for the field of reproduction and the application of assisted reproductive technologies (ART). Here, we have developed a platform to grow oviductal organoids from five domestic species-bovine, porcine, equine, feline, and canine. The organoids were grown progressively from single cells derived from the enzymatic digestion of freshly collected infundibular/fimbrial samples. The addition of WNT, TGFß, BMP, ROCK, and Notch signaling pathway activators or inhibitors to the organoid culture medium suggested remarkable conservation of the molecular signals involved in oviductal epithelial development and differentiation across species. The gross morphology of organoids from all the domestic species was initially similar. However, some differences in size, complexity, and growth rate were subsequently observed and described. After 21 days, well-defined and synchronized motile ciliated cells were observed in organoids. Histopathologically, oviductal organoids mimicked their respective native tissue. In summary, we have carried out a detailed cross-species comparison of oviductal organoids, which would be valuable in advancing our knowledge of oviduct physiology and, potentially, help in increasing the success of ART.
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Organoides , Mascotas , Humanos , Femenino , Animales , Gatos , Bovinos , Caballos , Perros , Porcinos , Granjas , Trompas Uterinas , Diferenciación CelularRESUMEN
In the mammalian vestibular periphery, electrical activation of the efferent vestibular system (EVS) has two effects on afferent activity: 1) it increases background afferent discharge and 2) decreases afferent sensitivity to rotational stimuli. Although the cellular mechanisms underlying these two contrasting afferent responses remain obscure, we postulated that the reduction in afferent sensitivity was attributed, in part, to the activation of α9- containing nicotinic acetylcholine (ACh) receptors (α9*nAChRs) and small-conductance potassium channels (SK) in vestibular type II hair cells, as demonstrated in the peripheral vestibular system of other vertebrates. To test this hypothesis, we examined the effects of the predominant EVS neurotransmitter ACh on vestibular type II hair cells from wild-type (wt) and α9-subunit nAChR knockout (α9-/-) mice. Immunostaining for choline acetyltransferase revealed there were no obvious gross morphological differences in the peripheral EVS innervation among any of these strains. ACh application onto wt type II hair cells, at resting potentials, produced a fast inward current followed by a slower outward current, resulting in membrane hyperpolarization and decreased membrane resistance. Hyperpolarization and decreased resistance were due to gating of SK channels. Consistent with activation of α9*nAChRs and SK channels, these ACh-sensitive currents were antagonized by the α9*nAChR blocker strychnine and SK blockers apamin and tamapin. Type II hair cells from α9-/- mice, however, failed to respond to ACh at all. These results confirm the critical importance of α9nAChRs in efferent modulation of mammalian type II vestibular hair cells. Application of exogenous ACh reduces electrical impedance, thereby decreasing type II hair cell sensitivity. NEW & NOTEWORTHY Expression of α9 nicotinic subunit was crucial for fast cholinergic modulation of mammalian vestibular type II hair cells. These findings show a multifaceted efferent mechanism for altering hair cell membrane potential and decreasing membrane resistance that should reduce sensitivity to hair bundle displacements.
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Acetilcolina/metabolismo , Células Ciliadas Vestibulares/metabolismo , Potenciales de la Membrana , Receptores Nicotínicos/metabolismo , Acetilcolina/farmacología , Animales , Apamina/farmacología , Femenino , Células Ciliadas Vestibulares/efectos de los fármacos , Células Ciliadas Vestibulares/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Bloqueadores de los Canales de Potasio/farmacología , Receptores Nicotínicos/genética , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/antagonistas & inhibidores , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo , Estricnina/farmacologíaRESUMEN
INTRODUCTION: The efferent vestibular system (EVS) is a feedback circuit thought to modulate vestibular afferent activity by inhibiting type II hair cells and exciting calyx-bearing afferents in the peripheral vestibular organs. In a previous study, we suggested EVS activity may contribute to the effects of motion sickness. To determine an association between motion sickness and EVS activity, we examined the effects of provocative motion (PM) on c-Fos expression in brainstem efferent vestibular nucleus (EVN) neurons that are the source of efferent innervation in the peripheral vestibular organs. METHODS: c-Fos is an immediate early gene product expressed in stimulated neurons and is a well-established marker of neuronal activation. To study the effects of PM, young adult C57/BL6 wild-type (WT), aged WT, and young adult transgenic Chat-gCaMP6f mice were exposed to PM, and tail temperature (Ttail ) was monitored using infrared imaging. After PM, we used immunohistochemistry to label EVN neurons to determine any changes in c-Fos expression. All tissue was imaged using laser scanning confocal microscopy. RESULTS: Infrared recording of Ttail during PM indicated that young adult WT and transgenic mice displayed a typical motion sickness response (tail warming), but not in aged WT mice. Similarly, brainstem EVN neurons showed increased expression of c-Fos protein after PM in young adult WT and transgenic mice but not in aged cohorts. CONCLUSION: We present evidence that motion sickness symptoms and increased activation of EVN neurons occur in young adult WT and transgenic mice in response to PM. In contrast, aged WT mice showed no signs of motion sickness and no change in c-Fos expression when exposed to the same provocative stimulus.
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Mareo por Movimiento , Ratones , Animales , Mareo por Movimiento/metabolismo , Neuronas/metabolismo , Núcleos Vestibulares/metabolismo , Neuronas Eferentes/metabolismo , Ratones TransgénicosRESUMEN
Cholinergic circuits in the central nervous system are vulnerable to age-related functional decline, but it is not known if aging impacts cholinergic signaling in the vestibular sensory organs, which are critically important to balance maintenance and visual gaze stability. We have previously shown cholinergic neurotransmission between vestibular efferent terminals and type II mechanosensory hair cells requires the alpha9 (Chrna9) nicotinic receptor subunit. Homozygous knockout of the alpha9 subunit causes vestibulo-ocular reflex adaptation deficits that mirror those observed in aged mice. This prompted examination of cholinergic signaling in the vestibular sensory organs of aged mice. We confirmed older (>24 months) mice had impaired performance in a balance beam task compared to young (3-4 months) adult mice. While there was no qualitative loss of cholinergic axon varicosities in the crista ampullaris of old mice, qPCR analysis revealed reduced expression of nicotinic receptor subunit genes Chrna1, Chrna9, and Chrna10 in the cristae of old relative to young mice. Functionally, single-cell patch clamp recordings taken from type II vestibular hair cells exposed to acetylcholine show reduced conductance through alpha9/10 subunit-containing nicotinic receptors in older mice, despite preserved passive membrane properties and voltage-activated conductances. These findings suggest that cholinergic signaling in the peripheral vestibular sensory organs is vulnerable to aging processes, manifesting in dynamic molecular and functional age-related changes. Given the importance of these organs to our everyday activities, and the dramatic increase in fall incidence in the older, further investigation into the mechanisms of altered peripheral vestibular function in older humans is warranted.
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Células Ciliadas Vestibulares , Receptores Nicotínicos , Vestíbulo del Laberinto , Humanos , Ratones , Animales , Anciano , Ratones Endogámicos C57BL , Vestíbulo del Laberinto/metabolismo , Células Ciliadas Vestibulares/metabolismo , Colinérgicos/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismoRESUMEN
The roles and connectivity of specific types of neurons within the spinal cord dorsal horn (DH) are being delineated at a rapid rate to provide an increasingly detailed view of the circuits underpinning spinal pain processing. However, the effects of these connections for broader network activity in the DH remain less well understood because most studies focus on the activity of single neurons and small microcircuits. Alternatively, the use of microelectrode arrays (MEAs), which can monitor electrical activity across many cells, provides high spatial and temporal resolution of neural activity. Here, the use of MEAs with mouse spinal cord slices to study DH activity induced by chemically stimulating DH circuits with 4-aminopyridine (4-AP) is described. The resulting rhythmic activity is restricted to the superficial DH, stable over time, blocked by tetrodotoxin, and can be investigated in different slice orientations. Together, this preparation provides a platform to investigate DH circuit activity in tissue from naïve animals, animal models of chronic pain, and mice with genetically altered nociceptive function. Furthermore, MEA recordings in 4-AP-stimulated spinal cord slices can be used as a rapid screening tool to assess the capacity of novel antinociceptive compounds to disrupt activity in the spinal cord DH.
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Nocicepción , Asta Dorsal de la Médula Espinal , 4-Aminopiridina , Animales , Ratones , Microelectrodos , Neuronas , Médula Espinal/fisiología , Asta Dorsal de la Médula Espinal/fisiologíaRESUMEN
Sensory neurons within DRGs are broadly divided into three types that transmit nociceptive, mechanical, and proprioceptive signals. These subtypes are established during in utero development when sensory neurons differentiate into distinct categories according to a complex developmental plan. Most of what we know about this developmental plan comes from studies in rodents and little is known about this process in humans. The present study documents the expression of key genes involved in human sensory neuron development during the late first and early second trimesters (9-16WG). We observed a decrease in the expression of SOX10 and BRN3A, factors associated with migration and proliferation of sensory neurons, towards the end of the first trimester. Small and large sensory neuron populations also emerged at the end of the first trimester, as well as the transcription factors responsible for defining distinct sensory neuron types. NTRK1, which is expressed in nociceptive neurons, emerged first at ~11 WG followed by NTRK2 in mechanoreceptors at ~12 WG, with NTRK3 for proprioceptors peaking at ~14 WG. These peaks were followed by increased expression of their respective neurotrophic factors. Our results show significant differences in the expression of key signalling molecules for human DRG development versus that of rodents, most notably the expression of neurotrophins that promote the survival of sensory neuron types. This highlights the importance of examining molecular changes in humans to better inform the application of data collected in pre-clinical models.
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Ganglios Espinales , Células Receptoras Sensoriales , Diferenciación Celular , Femenino , Desarrollo Fetal , Humanos , Embarazo , Segundo Trimestre del EmbarazoRESUMEN
The precise functional role of the Efferent Vestibular System (EVS) is still unclear, but the auditory olivocochlear efferent system has served as a reasonable model on the effects of a cholinergic and peptidergic input on inner ear organs. However, it is important to appreciate the similarities and differences in the structure of the two efferent systems, especially within the same animal model. Here, we examine the anatomy of the mouse EVS, from its central origin in the Efferent Vestibular Nucleus (EVN) of the brainstem, to its peripheral terminations in the vestibular organs, and we compare these findings to known mouse olivocochlear anatomy. Using transgenic mouse lines and two different tracing strategies, we examine central and peripheral anatomical patterning, as well as the anatomical pathway of EVS axons as they leave the mouse brainstem. We separately tag the left and right efferent vestibular nuclei (EVN) using Cre-dependent, adeno-associated virus (AAV)-mediated expression of fluorescent reporters to map their central trajectory and their peripheral terminal fields. We couple this with Fluro-Gold retrograde labeling to quantify the proportion of ipsi- and contralaterally projecting cholinergic efferent neurons. As in some other mammals, the mouse EVN comprises one group of neurons located dorsal to the facial genu, close to the vestibular nuclei complex (VNC). There is an average of just 53 EVN neurons with rich dendritic arborizations towards the VNC. The majority of EVN neurons, 55%, project to the contralateral eighth nerve, crossing the midline rostral to the EVN, and 32% project to the ipsilateral eighth nerve. The vestibular organs, therefore, receive bilateral EVN innervation, but without the distinctive zonal innervation patterns suggested in gerbil. Similar to gerbil, however, our data also suggest that individual EVN neurons do not project bilaterally in mice. Taken together, these data provide a detailed map of EVN neurons from the brainstem to the periphery and strong anatomical support for a dominant contralateral efferent innervation in mammals.
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Neuronas Eferentes , Vestíbulo del Laberinto , Animales , Tronco Encefálico , Vías Eferentes , Mamíferos , Ratones , Neuronas , Neuronas Eferentes/metabolismo , Núcleos VestibularesRESUMEN
Sodium channel expression in inner ear afferents is essential for the transmission of vestibular and auditory information to the central nervous system. During development, however, there is also a transient expression of Na+ channels in vestibular and auditory hair cells. Using qPCR analysis, we describe the expression of four Na+ channel genes, SCN5A (Nav1.5), SCN8A (Nav1.6), SCN9A (Nav1.7), and SCN10A (Nav1.8) in the human fetal cristae ampullares, utricle, and base, middle, and apex of the cochlea. Our data show distinct patterns of Na+ channel gene expression with age and between these inner ear organs. In the utricle, there was a general trend toward fold-change increases in expression of SCN8A, SCN9A, and SCN10A with age, while the crista exhibited fold-change increases in SCN5A and SCN8A and fold-change decreases in SCN9A and SCN10A. Fold-change differences of each gene in the cochlea were more complex and likely related to distinct patterns of expression based on tonotopy. Generally, the relative expression of SCN genes in the cochlea was greater than that in utricle and cristae ampullares. We also recorded Na+ currents from developing human vestibular hair cells aged 10-11 weeks gestation (WG), 12-13 WG, and 14+ WG and found there is a decrease in the number of vestibular hair cells that exhibit Na+ currents with increasing gestational age. Na+ current properties and responses to the application of tetrodotoxin (TTX; 1 µM) in human fetal vestibular hair cells are consistent with those recorded in other species during embryonic and postnatal development. Both TTX-sensitive and TTX-resistant currents are present in human fetal vestibular hair cells. These results provide a timeline of sodium channel gene expression in inner ear neuroepithelium and the physiological characterization of Na+ currents in human fetal vestibular neuroepithelium. Understanding the normal developmental timeline of ion channel gene expression and when cells express functional ion channels is essential information for regenerative technologies.
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The inner ear is a complex organ containing highly specialised cell types and structures that are critical for sensing sound and movement. In vivo, the inner ear is difficult to study due to the osseous nature of the otic capsule and its encapsulation within an intricate bony labyrinth. As such, mammalian inner ear explants are an invaluable tool for the study and manipulation of the complex intercellular connections, structures, and cell types within this specialised organ. The greatest strength of this technique is that the complete organ of Corti, or peripheral vestibular organs including hair cells, supporting cells and accompanying neurons, is maintained in its in situ form. The greatest weakness of in vitro hair cell preparations is the short time frame in which the explanted tissue remains viable. Yet, cochlear explants have proven to be an excellent experimental model for understanding the fundamental aspects of auditory biology, substantiated by their use for over 40 years. In this protocol, we present a modernised inner ear explant technique that employs organotypic cell culture inserts and serum free media. This approach decreases the likelihood of explant damage by eliminating the need for adhesive substances. Serum free media also restricts excessive cellular outgrowth and inter-experimental variability, both of which are side effects of exogenous serum addition to cell cultures. The protocol described can be applied to culture both cochlear and vestibular explants from various mammals. Example outcomes are demonstrated by immunohistochemistry, hair cell quantification, and electrophysiological recordings to validate the versatility and viability of the protocol.
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We present preliminary functional data from human vestibular hair cells and primary afferent calyx terminals during fetal development. Whole-cell recordings were obtained from hair cells or calyx terminals in semi-intact cristae prepared from human fetuses aged between 11 and 18 weeks gestation (WG). During early fetal development (11-14 WG), hair cells expressed whole-cell conductances that were qualitatively similar but quantitatively smaller than those observed previously in mature rodent type II hair cells. As development progressed (15-18 WG), peak outward conductances increased in putative type II hair cells but did not reach amplitudes observed in adult human hair cells. Type I hair cells express a specific low-voltage activating conductance, G K,L. A similar current was first observed at 15 WG but remained relatively small, even at 18 WG. The presence of a "collapsing" tail current indicates a maturing type I hair cell phenotype and suggests the presence of a surrounding calyx afferent terminal. We were also able to record from calyx afferent terminals in 15-18 WG cristae. In voltage clamp, these terminals exhibited fast inactivating inward as well as slower outward conductances, and in current clamp, discharged a single action potential during depolarizing steps. Together, these data suggest the major functional characteristics of type I and type II hair cells and calyx terminals are present by 18 WG. Our study also describes a new preparation for the functional investigation of key events that occur during maturation of human vestibular organs.