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[hal-04884151] Automatic ovarian follicle detection using object detection models
Ovaries are of paramount importance in reproduction as they produce female gametes through a complex developmental process known as folliculogenesis. In the prospect of better understanding the mechanisms of folliculogenesis and of developing novel pharmacological approaches to control it, it is important to accurately and quantitatively assess the later stages of ovarian folliculogenesis (i.e. the formation of antral follicles and corpus lutea). Manual counting from histological sections is commonly employed to determine the number of these follicular structures, however it is a laborious and error prone task. In this work, we show the benefits of deep learning models for counting antral follicles and corpus lutea in ovarian histology sections. Here, we use various backbone architectures to build two one-stage object detection models, i.e. YOLO and RetinaNet. We employ transfer learning, early stopping, and data augmentation approaches to improve the generalizability of the object detectors. Furthermore, we use sampling strategy to mitigate the foreground-foreground class imbalance and focal loss to reduce the imbalance between the foreground-background classes. Our models were trained and validated using a dataset containing only 1000 images. With RetinaNet, we achieved a mean average precision of 83% whereas with YOLO of 75% on the testing dataset. Our results demonstrate that deep learning methods are useful to speed up the follicle counting process and improve accuracy by correcting manual counting errors.
ano.nymous@ccsd.cnrs.fr.invalid (Maya Haj Hassan) 25 Feb 2025
https://hal.inrae.fr/hal-04884151v1
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[hal-04785377] Effect of the social environment on olfaction and social skills in wild-type and a mouse model of autism
Autism spectrum disorders (ASD) are complex, polygenic and heterogenous neurodevelopmental conditions. The severity of autism-associated variants is influenced by environmental factors, particularly social experiences during the critical neurodevelopmental period. While early behavioral interventions have shown efficacy in some children with autism, pharmacological support for core features - impairments in social interaction and communication, and stereotyped or restricted behaviors - is currently lacking. In this study, we examined how the social environment influences both wild-type (WT) and Shank3 knockout (KO) mice, a model reflecting core autism-like traits. Our findings revealed that chronic social isolation enhanced social interaction and olfactory neuron responses in WT animals. Furthermore, it restored impairments in social novelty preference and olfactory function, as well as self-grooming in Shank3 KO mice. Conversely, an enriched social environment heightened social interest toward novel conspecifics in WT mice, but elicited the opposite effect in Shank3 KO mice. Notably, Shank3 KO mice displayed distinct social responses when exposed to WT or Shank3 KO mice. These results offer novel insights that could favor the implementation of behavioral interventions and inclusive classroom programs for children with ASD.
ano.nymous@ccsd.cnrs.fr.invalid (Caroline Gora) 18 Nov 2024
https://hal.inrae.fr/hal-04785377v1
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[hal-04777090] Oviduct epithelial spheroids during in vitro culture of bovine embryos mitigate oxidative stress, improve blastocyst quality and change the embryonic transcriptome
Background In vitro embryo production is increasingly used for genetic improvement in cattle but bypasses the oviduct environment and exposes the embryos to oxidative stress with deleterious effects on further development. Here we aimed to examine the effect of oviduct epithelial spheroids (OES) on embryo development and quality in terms of morphology and gene expression during two co-culture times (4 days: up to embryonic genome activation at 8–16 cell stage vs. 7 days: up to blastocyst stage) and under two oxygen levels (5% vs. 20%). Methods Bovine presumptive zygotes produced by in vitro fertilization (day 0) using in-vitro matured oocytes were cultured in droplets of synthetic oviductal fluid (SOF) medium with or without (controls) OES for 4 or 7 days under 5% or 20% oxygen (4 treated and 2 control groups). Cleavage rates were evaluated on day 2 and blastocyst rates on days 7–8. Expanded blastocysts on days 7–8 were evaluated for total cell numbers and gene expression analysis by RNA-sequencing. Results Under 20% oxygen, blastocyst rates and total cell numbers were significantly higher in the presence of OES for 4 and 7 days compared to controls ( P < 0.05), with no difference according to the co-culture time. Under 5% oxygen, the presence of OES did not affect blastocyst rates but increased the number of cells per blastocyst after 7 days of co-culture ( P < 0.05). Both oxygen level and OES co-culture had a significant impact on the embryonic transcriptome. The highest number of differentially expressed genes (DEGs) was identified after 7 days of co-culture under 20% oxygen. DEGs were involved in a wide range of functions, including lipid metabolism, membrane organization, response to external signals, early embryo development, and transport of small molecules among the most significantly impacted. Conclusion OES had beneficial effects on embryo development and quality under both 5% and 20% oxygen, mitigating oxidative stress. Stronger effects on embryo quality and transcriptome were obtained after 7 than 4 days of co-culture. This study shows the impact of OES on embryo development and reveals potential molecular targets of OES-embryo dialog involved in response to stress and early embryonic development.
ano.nymous@ccsd.cnrs.fr.invalid (Thanya Pranomphon) 12 Nov 2024
https://hal.inrae.fr/hal-04777090v1
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[hal-04735026] Exploring neuronal markers and early social environment influence in divergent quail lines selected for social motivation
Many species, including humans exhibit a wide range of social behaviors that are crucial for the adaptation and survival of most species. Brain organization and function are shaped by genetic and environmental factors, although their precise contributions have been relatively understudied in the context of artificial selection. We used divergent lines of quail selected on their high versus low level of motivation to approach a group of conspecifics (S + and S-, respectively) to investigate the influence of genetic selection and early social environment on sociability. We observed distinct sex-and brainregion-specific expression patterns of three neuronal markers: mesotocin, and vasotocin, the avian homologues of mammalian oxytocin and vasopressin, as well as aromatase, the enzyme that converts androgens into estrogens. These markers displayed pronounced and neuroanatomically specific differences between S + and S-quail. Additionally, in a second experiment, we assessed the influence of early social environment on social skills in juvenile birds. Mixing S + and S-resulted in more S-males approaching the group without affecting the sociability of S + or other behaviors, suggesting that the early social environment may influence the results of genetic selection. In conclusion, the divergent quail lines offer a valuable model for unraveling the neuronal and behavioral mechanisms underlying social behaviors.
ano.nymous@ccsd.cnrs.fr.invalid (Lucas Court) 14 Oct 2024
https://hal.science/hal-04735026v1
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[hal-04785629] Social experience is associated with a differential role of aromatase neurons in sexual behavior and territorial aggression in male mice
Aromatase (Aro+) neurons located in the Bed Nucleus of the Stria Terminalis (BNST) are crucial for the display of both sexual behavior and territorial aggression in naive male mice. However, little is known about how Aro + neuron circuitry is influenced by social experience. Using a combination of chemogenetics, activity mapping and retrograde viral tracing we show that social experience modulates Aro + neurons during sexual behavior and territorial aggression. Chemogenetic inhibition of BNST Aro + neurons in socially experienced male mice revealed that these neurons are required for territorial aggression, but not for sexual behavior. Behavior testing in experienced animals showed a specific increase in activation in the vomeronasal organ (VNO) and the Medial Amygdala (MeA) after sexual behavior but not territorial aggression, assessed by Egr1 expression. We also observed an increase of Egr1 cells in the medial Preoptic Area (mPOA), a brain region implicated in the display of sexual behavior. Combined retrograde viral tracing and Egr1 immunodetection showed that a subset of the activated cells in the MeA are Aro + neurons projecting to the mPOA. These results highlight that social experience induces a differential neural activity in the circuitry controlling sexual behavior and aggression, which include MeA Aro + neurons projecting to the mPOA.
ano.nymous@ccsd.cnrs.fr.invalid (Elliott Trives) 15 Nov 2024
https://hal.science/hal-04785629v1
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[hal-04668790] Facial blushing and feather fluffing are indicators of emotions in domestic fowl (Gallus gallus domesticus)
The study of facial expressions in mammals provided great advances in the identification of their emotions and then in the comprehension of their sentience. So far, this area of research has excluded birds. With a naturalist approach, we analysed facial blushing and feather displays in domestic fowl. Hens were filmed in situations contrasting in emotional valence and arousal level: situations known to indicate calm states (positive valence / low arousal), have rewarding effects (positive valence / high arousal) or induce fear-related behaviour (negative valence / high arousal). Head feather position as well as skin redness of comb, wattles, ear lobes and cheeks varied across these situations. Skin of all four areas was less red in situations with low arousal compared to situations with higher arousal. Furthermore, skin redness of the cheeks and ear lobes also varied depending on the valence of the situation: redness was higher in situations with negative valence compared to situations with positive valence. Feather position also varied with the situations. Feather fluffing was mostly observed in positively valenced situations, except when hens were eating. We conclude that hens have facial displays that reveal their emotions and that blushing is not exclusive to humans. This opens a promising way to explore the emotional lives of birds, which is a critical step when trying to improve poultry welfare.
ano.nymous@ccsd.cnrs.fr.invalid (Cécile Arnould) 29 Nov 2024
https://hal.science/hal-04668790v1
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[hal-04669483] Automatic ovarian follicle detection using object detection models
Ovaries are of paramount importance in reproduction as they produce female gametes through a complex developmental process known as folliculogenesis. In the prospect of better understanding the mechanisms of folliculogenesis and of developing novel pharmacological approaches to control it, it is important to accurately and quantitatively assess the later stages of ovarian folliculogenesis (i.e. the formation of antral follicles and corpus lutea). Manual counting from histological sections is commonly employed to determine the number of these follicular structures, however it is a laborious and error prone task. In this work, we show the benefits of deep learning models for counting antral follicles and corpus lutea in ovarian histology sections. Here, we use various backbone architectures to build two one-stage object detection models, i.e. YOLO and RetinaNet. We employ transfer learning, early stopping, and data augmentation approaches to improve the generalizability of the object detectors. Furthermore, we use sampling strategy to mitigate the foreground-foreground class imbalance and focal loss to reduce the imbalance between the foreground-background classes. Our models were trained and validated using a dataset containing only 1000 images. With RetinaNet, we achieved a mean average precision of 83\% whereas with YOLO of 75% on the testing dataset. Our results demonstrate that deep learning methods are useful to speed up the follicle counting process and improve accuracy by correcting manual counting errors.
ano.nymous@ccsd.cnrs.fr.invalid (Maya Haj Hassan) 08 Aug 2024
https://hal.science/hal-04669483v1
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[hal-04669329] Basal ewe folliculogenesis from preantral to antral stages: Gene clustering and Connexin 43 protein profile by transparisation coupled to immunolocalization
Background: Basal follicular growth is a slow process controlled by a privileged molecular dialogue between the oocyte and somatic cells. A pivotal event is the formation of an antral cavity within the granulosa and the differentiation of the oocyte’s closest cells into cumulus cells. Towards elucidating the mechanisms regulating these morphological and functional changes, we have analyzed expression of a panel of genes from the small preantral to the small antral follicular stages. Next, we have focused onto Connexin43 encoded by the GJA1 gene. Connexins are components of gap-junctions, channels that form between neighbour granulosa/cumulus cells, but also between transzonal projections (TZP) and the oocyte. The exchange of small molecules through gap-junctions throughout folliculogenesis is crucial for ultimately producing a mature oocyte. Methods: 1389 follicles (80-800 µm) were collected from ovarian cortex strips of prepubertal ewes, and dispatched in 43 samples representing 5 follicular size classes (small, medium and large preantral, early and small antral). Expression of 40 genes was analyzed using microfluidic qPCR followed by hierarchical clustering. Protein expression was examined after tissue clearing by immunofluorescence. Results: Hierarchical clustering segregated preantral and antral follicles and revealed changes in the expression of genes involved in cell proliferation and differentiation including hormone secretion, but also in the communication between the oocyte, cumulus and granulosa cells. GJA1 increased slightly during follicle growth. Connexin43 was detected between granulosa/cumulus cells and at the periphery of the zona pellucida. Conclusions: The destiny of a follicle relies on a delicate balance in the expression and activity of factors involved in proliferation, differentiation, apoptosis and cell-cell communication. Evaluating these factors in in vivo developed follicles sets the ground for validating biotechnologies of in vitro follicle culture, which aim at producing an oocyte of good quality for subsequent fertilization in the context of fertility preservation. Conflict of interest: The authors have no conflicts of interest to disclose.
ano.nymous@ccsd.cnrs.fr.invalid (Véronique Cadoret) 09 Aug 2024
https://hal.inrae.fr/hal-04669329v3
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[hal-04574339] Exploration of skin redness and immunoglobulin A as markers of the affective states of hens
Non-invasive markers of affective states can help understanding animals' perception of situations and improving their welfare. These markers are scarce in avian species. In this study, we investigate the potential relation between alterations in facial skin redness in hens and their corresponding affective states. Six hens were filmed in both naturally unfolding scenarios and controlled tests designed to elicit various affective states. The facial skin redness was measured from images extracted from the videos. Our observations revealed that hens exhibited the highest degree of facial skin redness in negative situations of high arousal, a high redness in positive situations of high arousal, and the lowest in positive situations of low arousal. In a second study, we further examined whether facial skin redness and secretory immunoglobulin A (S-IgA) can serve as markers for the quality of the human-animal relationship. Two groups of hens, one habituated to humans (n=13) and one non-habituated (n=12), were compared for general fearfulness in an open field test and for fear of humans in a reactivity to human test. In the open-field test, there were no statistical differences in general fearfulness, facial skin redness or S-IgA concentrations between both groups. However, habituated hens exhibited significantly lower fearfulness and facial skin redness in the presence of humans compared to non-habituated hens in the reactivity to human test. Additionally, habituated hens showed significant lower S-IgA concentration in lachrymal fluid in the presence of humans, with no significant differences in saliva or cloacal samples. We propose that changes in facial skin redness reflect variations in affective states and can be used as a marker for assessing the quality of the human-hen relationship. The relationship between S-IgA concentrations and affective states requires further investigation.
ano.nymous@ccsd.cnrs.fr.invalid (Delphine Soulet) 14 May 2024
https://hal.inrae.fr/hal-04574339v1
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[hal-04667755] Hair from sexually active bucks strongly activates olfactory sensory inputs but fails to trigger early first ovulation in prepubescent does
Early exposure of does to sexually active bucks triggers early puberty onset correlating with neuroendocrine changes. However, the sensory pathways that are stimulated by the male are still unknown. Here, we assessed whether responses to olfactory stimuli are modulated by social experience (exposure to males or not) and/or endocrine status (prepubescent or pubescent). We used a calcium imaging approach on goat sensory cells from the main olfactory epithelium (MOE) and the vomeronasal organ (VNO). For both cell types, we observed robust responses to active male hair in females under three physiological conditions: prepubescent females isolated from males (ISOL PrePub), pubescent females exposed to males (INT Pub) and isolated females (ISOL Pub). Response analysis showed overall greater proportion of responses to buck hair in ISOL PrePub. We hypothesized that females would be more responsive to active buck hair during the prepubertal period, with numerous responses perhaps originating from immature neurons. We also observed a greater proportion of mature olfactory neurons in the MOE and VNO of INT Pub females suggesting that male exposure can induce plastic changes on olfactory cell function and organization. To determine whether stimulation by male odor can advance puberty, we exposed prepubescent does to active buck hair (ODOR). In both ODOR and females isolated from males (ISOL) groups, puberty was reached one month after females exposed to intact bucks (INT), suggesting that olfactory stimulation is not sufficient to trigger puberty.
ano.nymous@ccsd.cnrs.fr.invalid (Maxime Meunier) 21 Nov 2024
https://hal.inrae.fr/hal-04667755v1
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[hal-04514599] Sperm binding to oviduct epithelial spheroids varies among males and ejaculates but not among females in pigs
The elimination of ejaculates and males with low fertility despite good sperm motility and morphology is crucial to maintain high pregnancy rates after artificial insemination (AI) in farm animals. The ability of sperm to survive in the female tract is particularly crucial in pigs due to the large variation in the timing between AI and ovulation and the high number of oocytes to fertilise. The objective of this study was to characterise a new in vitro model of oviduct sperm reservoir using porcine oviduct epithelial spheroids (OES) and to assess the variability in sperm binding to OES among gilts, boars and their ejaculates. Isthmic mucosa fragments were collected from gilt oviducts at a slaughterhouse, and after 48 h of culture, the OES that had spontaneously formed were sorted according to their vesicle shape and size (150-200 mu m in diameter) for characterisation and sperm binding assays. The OES contained viable, cytokeratin-positive and vimentin-negative cells, of which 36.4 +/- 2.0% were multiciliated. The average proportion of multiciliated cells per OES did not change among culture replicates. After co-incubation with boar fresh semen, only sperm of normal morphology were found to bind, by their head, to cilia of OES. The density of sperm bound to the OES surface increased linearly with sperm concentration. The bound sperm density on OES was used to assess the binding capacity of fresh ejaculates collected from Pietrain boars. For a given ejaculate, the bound sperm density did not vary among pools of OES female donors. The analysis of five successive ejaculates from nine boars indicated significant differences in bound sperm densities on the OES among individual boars and their ejaculates (P < 0.01). There was no correlation between the sperm bound density and sperm parameters measured by computer-assisted sperm analysis or the initial dilution of the ejaculate. In conclusion, the OES characterised in this study offered physiological conditions to study sperm binding to the isthmic reservoir and evidenced that sperm from different ejaculates and different boars vary in their ability to bind to these oviduct spheroids despite homogeneous motility and morphology.
ano.nymous@ccsd.cnrs.fr.invalid (Lorraine Schmaltz) 21 Mar 2024
https://hal.inrae.fr/hal-04514599v1
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[hal-04408256] CoNIC Challenge: Pushing the frontiers of nuclear detection, segmentation, classification and counting
Nuclear detection, segmentation and morphometric profiling are essential in helping us further understand the relationship between histology and patient outcome. To drive innovation in this area, we setup a community-wide challenge using the largest available dataset of its kind to assess nuclear segmentation and cellular composition. Our challenge, named CoNIC, stimulated the development of reproducible algorithms for cellular recognition with real-time result inspection on public leaderboards. We conducted an extensive post-challenge analysis based on the top-performing models using 1,658 whole-slide images of colon tissue. With around 700 million detected nuclei per model, associated features were used for dysplasia grading and survival analysis, where we demonstrated that the challenge's improvement over the previous state-of-the-art led to significant boosts in downstream performance. Our findings also suggest that eosinophils and neutrophils play an important role in the tumour microevironment. We release challenge models and WSI-level results to foster the development of further methods for biomarker discovery.
ano.nymous@ccsd.cnrs.fr.invalid (Simon Graham) 15 Oct 2024
https://hal.science/hal-04408256v1
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[hal-04514574] Characterization of oviduct epithelial spheroids for the study of embryo–maternal communication in cattle
Most in vitro models of oviduct epithelial cells (OEC) used thus far to gain insights into embryo–maternal communication induce cell dedifferentiation or are technically challenging. Moreover, although the presence of developing embryos has been shown to alter gene expression in OEC, the effect of embryos on OEC physiology remains largely unknown. Here, we propose a model based on bovine oviduct epithelial spheroids (OES) with specific shape and diameter (100–200 μm) criteria. The aims of this study were to i) determine the appropriate culture conditions of bovine OES cultured in suspension by evaluating their morphology, total cell number, viability, and activity of ciliated cells; ii) monitor gene expression in OES at the time of their formation (day 0) and over the 10 days of culture; and iii) test whether the vicinity of developing embryos affects OES quality criteria. On day 10, the proportions of vesicle-shaped OES (V-OES) were higher in M199/500 (500 μl of HEPES-buffered TCM-199) and synthetic oviduct fluid (SOF)/25 (25-μL droplet of SOF medium under mineral oil) than in M199/25 (25-μL droplet of M199 under mineral oil). The proportion of viable cells in V-OES was not affected by culture conditions and remained high (>80%) through day 10. The total number of cells per V-OES decreased over time except in SOF/25, while the proportions of ciliated cells increased over time in M199/500 but decreased in M199/25 and SOF/25. The movement amplitude of OES in suspension decreased over time under all culture conditions. Moreover, the gene expression of ANXA1, ESR1, HSPA8, and HSPA1A in OES remained stable during culture, while that of PGR and OVGP1 decreased from day 0 to day 10. Last, the co-culture of developing embryos with OES in SOF/25 increased the rates of blastocysts on days 7 and 8 compared to embryos cultured alone, and increased the proportion of V-OES compared to OES cultured alone. In conclusion, M199/500 and SOF/25 provided the optimal conditions for the long-time culture of OES. The supporting effect of OES on embryo development and of developing embryos on OES morphology was evidenced for the first time. Altogether, these results point OES as an easy-to-use, standardizable, and physiological model to study embryo–maternal interactions in cattle.
ano.nymous@ccsd.cnrs.fr.invalid (Thanya Pranomphon) 21 Mar 2024
https://hal.inrae.fr/hal-04514574v1
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[hal-04158294] Sensing and avoiding sick conspecifics requires Gαi2+ vomeronasal neurons
Abstract Background Rodents utilize chemical cues to recognize and avoid other conspecifics infected with pathogens. Infection with pathogens and acute inflammation alter the repertoire and signature of olfactory stimuli emitted by a sick individual. These cues are recognized by healthy conspecifics via the vomeronasal or accessory olfactory system, triggering an innate form of avoidance behavior. However, the molecular identity of the sensory neurons and the higher neural circuits involved in the detection of sick conspecifics remain poorly understood. Results We employed mice that are in an acute state of inflammation induced by systemic administration of lipopolysaccharide (LPS). Through conditional knockout of the G-protein Gαi2 and deletion of other key sensory transduction molecules (Trpc2 and a cluster of 16 vomeronasal type 1 receptors), in combination with behavioral testing, subcellular Ca 2+ imaging, and pS6 and c-Fos neuronal activity mapping in freely behaving mice, we show that the Gαi2 + vomeronasal subsystem is required for the detection and avoidance of LPS-treated mice. The active components underlying this avoidance are contained in urine whereas feces extract and two selected bile acids, although detected in a Gαi2-dependent manner, failed to evoke avoidance behavior. Our analyses of dendritic Ca 2+ responses in vomeronasal sensory neurons provide insight into the discrimination capabilities of these neurons for urine fractions from LPS-treated mice, and how this discrimination depends on Gαi2. We observed Gαi2-dependent stimulation of multiple brain areas including medial amygdala, ventromedial hypothalamus, and periaqueductal grey. We also identified the lateral habenula, a brain region implicated in negative reward prediction in aversive learning, as a previously unknown target involved in these tasks. Conclusions Our physiological and behavioral analyses indicate that the sensing and avoidance of LPS-treated sick conspecifics depend on the Gαi2 vomeronasal subsystem. Our observations point to a central role of brain circuits downstream of the olfactory periphery and in the lateral habenula in the detection and avoidance of sick conspecifics, providing new insights into the neural substrates and circuit logic of the sensing of inflammation in mice.
ano.nymous@ccsd.cnrs.fr.invalid (Jan Weiss) 11 Jul 2023
https://hal.inrae.fr/hal-04158294v1
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[hal-04233205] Seasonal remodeling of the progenitor pool and its distribution in the ewe mediobasal hypothalamus
Recent studies have reported the presence of adult neurogenesis in the arcuate nucleus periventricular space (pvARH) and in the median eminence (ME), two structures involved in reproductive function. In sheep, a seasonal mammal, decreasing daylight in autumn induces a higher neurogenic activity in these two structures. However, the different types of neural stem and progenitor cells (NSCs/NPCs) that populate the arcuate nucleus and median eminence, as well as their location, have not been evaluated. Here, using semi-automatic image analyzing processes, we identified and quantified the different populations of NSCs/NPCs, showing that, during short days, higher densities of [SOX2 +] cells are found in pvARH and ME. In the pvARH, higher densities of astrocytic and oligodendrocitic progenitors mainly contribute to these variations. The different populations of NSCs/NPCs were mapped according to their position relative to the third ventricle and their proximity to the vasculature. We showed that [SOX2 +] cells extended deeper into the hypothalamic parenchyma during short days. Similarly, [SOX2 +] cells were found further from the vasculature in the pvARH and the ME, at this time of year, indicating the existence of migratory signals. The expression levels of neuregulin transcripts (NRGs), whose proteins are known to stimulate proliferation and adult neurogenesis and to regulate progenitor migration, as well as the expression levels of ERBB mRNAs, cognate receptors for NRGs, were assessed. We showed that mRNA expression changed seasonally in pvARH and ME, suggesting that the ErbB-NRG system is potentially involved in the photoperiodic regulation of neurogenesis in seasonal adult mammals.
ano.nymous@ccsd.cnrs.fr.invalid (Pierre-Marie Chevillard) 09 Oct 2023
https://hal.inrae.fr/hal-04233205v1
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[hal-04230035] 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.
ano.nymous@ccsd.cnrs.fr.invalid (Maxime A Meunier) 05 Oct 2023
https://cnrs.hal.science/hal-04230035v1
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[hal-04168459] Captive Blue-and-yellow macaws (Ara ararauna) show facial indicators of positive affect when reunited with their caregiver
In mammals, human-animal bonding is recognized as a source of positive affect for companion or farm animals. Because this remains unexplored in birds, we investigated captive parrots’ perspective of the human-animal relationship. We used a classical separation-reunion paradigm and predicted that variations in parrots’ facial displays and behaviours would indicate their appraisal of the relationship. The test was divided into three phases of two minutes each: the bird was placed in an unfamiliar environment with a familiar caregiver (union), then the bird was left alone (separation) and finally, the caregiver returned (reunion). The test was repeated 10 times for each bird and video recorded in order to analyze their behaviour. The data show significantly higher crown and nape feather heights, higher redness of the skin and higher frequency of contact-seeking behaviours during the union and reunion phases than during the separation phase during which they expressed long distance contact calls. We observed the expression of eye pinning during the union and reunion phases in one out of five macaws. We argue that variation in facial displays provides indicators of parrot’s positive appraisal of the caretaker presence. Our results broaden the scope for further studies on parrots’ expression of their subjective feelings.
ano.nymous@ccsd.cnrs.fr.invalid (Aline Bertin) 17 Nov 2023
https://hal.inrae.fr/hal-04168459v2
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[hal-04013883] Interactions between β-endorphin and kisspeptin neurons of the ewe arcuate nucleus are modulated by photoperiod
Opioid peptides are well-known modulators of the central control of reproduction. Among them, dynorphin coexpressed in kisspeptin (KP) neurons of the arcuate nucleus (ARC) has been thoroughly studied for its autocrine effect on KP release through κ opioid receptors. Other studies have suggested a role for β-endorphin (BEND), a peptide cleaved from the proopiomelanocortin (POMC) precursor, on food intake and central control of reproduction. Similarly to KP, BEND content in the ARC of sheep is modulated by day length and BEND modulates food intake in a dose-dependent manner. As KP levels in the ARC vary with photoperiodic and metabolic status, a photoperiod-driven influence of BEND neurons on neighboring KP neurons is plausible. The aim of this study was therefore to investigate a possible modulatory action of BEND on KP neurons located in the ovine ARC. Using confocal microscopy, numerous KP appositions on BEND neurons were found but there was no photoperiodic variation in the number of these interactions in ovariectomized, estradiol-replaced ewes. In contrast, BEND terminals on KP neurons were twice as numerous under short days (SD), in ewes having an activated gonadotropic axis, as compared to anestrus ewes under long days (LD). Injection of 5µg BEND into the third ventricle of SD ewes induced a significant and specific increase of activated KP neurons (16% versus 9% in controls) while the percentage of overall activated (c-Fos positive) neurons, was similar between both groups. These data suggest a photoperiod-dependent influence of BEND on KP neurons of the ARC, which may influence GnRH pulsatile secretion and inform KP neurons on the metabolic status.
ano.nymous@ccsd.cnrs.fr.invalid (Vincent Hellier) 07 Mar 2023
https://hal.science/hal-04013883v1
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[hal-04185480] Short photoperiod modulates behavior, cognition and hippocampal neurogenesis in male Japanese quail
The mechanisms underlying the photoperiodic control of reproduction in mammals and birds have been recently clarified. In contrast, the potential impact of photoperiod on more complex, integrative processes, such as cognitive behaviors, remains poorly characterized. Here, we investigated the impact of contrasted long and short photoperiods (LP, 16 h light/day and SP, 8 h light/day, respectively) on learning, spatial orientation abilities, and emotional reactivity in male Japanese quail. In addition, we quantified cell proliferation and young cell maturation/migration within the hippocampus, a brain region involved in spatial orientation. Our study reveals that, in male quail, SP increases emotional responses and spatial orientation abilities, compared to LP. Behaviorally, SP birds were found to be more fearful than LP birds, exhibiting more freezing in the open field and taking longer to exit the dark compartment in the emergence test. Furthermore, SP birds were significantly less aggressive than LP birds in a mirror test. Cognitively, SP birds were slower to habituate and learn a spatial orientation task compared to LP birds. However, during a recall test, SP birds performed better than LP birds. From a neuroanatomical standpoint, SP birds had a significantly lower density of young neurons, and also tended to have a lower density of mature neurons within the hippocampus, compared to LP birds. In conclusion, our data reveal that, beyond breeding control, photoperiod also exerts a profound influence on behavior, cognition, and brain plasticity, which comprise the seasonal program of this species.
ano.nymous@ccsd.cnrs.fr.invalid (Marion Georgelin) 22 Aug 2023
https://hal.inrae.fr/hal-04185480v1
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[hal-04178186] Spatial distribution of different lipids by mass spectrometry imaging may reveal signatures of follicular differentiation in ovine ovary
BACKGROUND-AIM Lipid metabolism is involved in the regulation of ovarian follicle growth, capacity to ovulate, corpus luteum (CL) formation, and luteolysis. Each cycle, from many antral follicles entering final growth, only dominant one can ovulate, others go through atresia. We aimed to map lipids through the whole ovary to explore their distribution according to follicle stage and the presence of blood vessels. METHODS Paraformaldehyde-fixed ovine ovaries were consecutively analyzed by ex vivo 3D Magnetic Resonance Imaging (MRI) and 2D/3D Mass Spectrometry Imaging (MSI). The whole organs were cut using cryostat to generate 10 μmthick sections spaced with intervals around 100 μm. Slides were coated with DHB matrix using M5 sprayer (HTX Technologies). MSI measurements were done on a RapifleX MALDI-TOF spectrometer (Bruker) in positive ion mode with m/z 100-1200 to detect lipids, with a lateral resolution of 30-40 μm. MSI data were treated by SCiLS Lab software. Immunohistochemistry was performed on adjacent sections, and 20x light microscopy images were acquired using the AxioScanZ1 scanner (Zeiss). RESULTS From two ovaries, 72 and 123 MSI sequences were acquired and analyzed separately. From each section, 180-300 ion density maps were generated, revealing different ions enriched in either follicular cells, or fluid, or blood vessels, or CL. After hierarchical clustering, segmentation maps clearly discriminated blood vessels, CL, and cortex/ interstitial tissue from antral follicles, due to specific lipid profiles. Then, for each ovary, all sections were aligned, merged into a 3D data set, in order to topography lipids between the follicles of different sizes and atretic stages. 3D representations allowed in situ cartography of blood vessels and reconstruction of dominant and subordinated follicles by specific molecular signatures of follicular cells and fluids. In addition, a multimodal analysis combining immunodetection of Hemoglobin subunit A and Proliferating Cell Nuclear Antigen confirmed lipid mapping to definite ovarian structures. CONCLUSIONS 2D and 3D-dimensional MSI of lipids allowed the spatial position of the follicles relative to blood flow and discrimination of follicular compartments that may help to enlighten the involvement of lipids in follicle differentiation.
ano.nymous@ccsd.cnrs.fr.invalid (Emilie Maugrion) 07 Aug 2023
https://hal.inrae.fr/hal-04178186v1
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[hal-04178194] Ovine ovary multimodal imaging combining ex vivo 3D magnetic resonance imaging, 2D histology and mass spectrometry imaging to study terminal folliculogenesis
BACKGROUND In ruminants, numerous antral follicles enter in final growth but only a dominant follicle will ovulate. Inside the follicle, follicular fluid (FF) and follicular cells have specific lipid contents, which change during follicular growth due to differential lipid uptake from blood and fatty acid metabolism of follicular cells. The objective was to develop a multimodal imaging approach to explore lipid distribution through the whole ovary. METHODS Ewe ovaries (Ovis aries) were fixed by 4% paraformaldehyde and analyzed using 3 Teslas Magnetic Resonance Imaging (MRI) instrument (Siemens). 3D images were acquired with an isotropic voxel size of 0.25 mm. 10 µm cryosections of the ovaries were analysed by Mass Spectrometry Imaging (MSI) using RapifleX MALDI-TOF spectrometer (Bruker). The MSI sequences were acquired with a spatial resolution of 30 µm/pixel, in positive ion mode to detect lipids in 100-1500 m/z range. Light microscopy 20x images of either hematoxylin, or Oil red stained sections were acquired using the AxioScanZ1 scanner (Zeiss). RESULTS MRI was performed on 28 whole ovaries. 15-71 antral follicles per ovary with inner diameter ranging from 0.25 mm to 10.5 mm were detected. Segmentation of 3D MRI images allowed measuring of FF volume in each follicle and determining their position. Five organs were sectioned, and either MSI of lipids, or histological staining were performed on ovary sections. Hierarchical clustering of lipid spectra, which discriminated antral follicles from blood vessels, luteal bodies and cortex, generated MSI segmentation 2D maps. Hematoxylin staining revealed the morphology of ovary sections. Lipid accumulation sites were detected by Oil red staining. Single 2D histological images were then aligned to MSI of adjacent sections. Both could be integrated within the 3D MRI ovary volume. By such 2.5D representations, the topology of lipids through ovarian compartments, or between the follicles of different sizes could be analyzed. CONCLUSIONS The combination of 3D MRI to optical microscopy and to 2D MSI has allowed access to new anatomical and structural information within the whole ovary, with especially a more precise mapping of lipids, enlightening their involvement in follicle differentiation through terminal folliculogenesis.
ano.nymous@ccsd.cnrs.fr.invalid (Hans Adriaensen) 07 Aug 2023
https://hal.inrae.fr/hal-04178194v1
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[hal-04179968] Développement de l’IMAGerie multimodale 3D : application à l’Ovaire
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ano.nymous@ccsd.cnrs.fr.invalid (Svetlana Uzbekova) 10 Aug 2023
https://hal.inrae.fr/hal-04179968v1
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[hal-03746290] Seasonal vascular plasticity in the mediobasal hypothalamus of the adult ewe
Sheep, like most seasonal mammals, exhibit a cyclic adaptive reproductive physiology that allows ewes to give birth to their progeny during the spring when environmental conditions are favorable to their survival. This process relies on the detection of day length (or photoperiod) and is associated with profound changes in cellular plasticity and gene expression in the hypothalamic-pituitary-gonadal axis, mechanisms that are suggested to participate in the seasonal adaptation of neuroendocrine circuits. Recently, pituitary vascular growth has been proposed as a seasonally regulated process in which the vascular endothelial growth factor A (VEGFA), a well-known angiogenic cytokine, is suspected to play a crucial role. However, whether this mechanism is restricted to the pituitary gland or also occurs in the mediobasal hypothalamus (MBH), a crucial contributor to the control of the reproductive function, remains unexplored. Using newly developed image analysis tools, we showed that the arcuate nucleus (ARH) of the MBH exhibits an enhanced vascular density during the long photoperiod or non-breeding season, associated with higher expression of VEGFA. In the median eminence (ME), a structure connecting the MBH to the pituitary gland, higher VEGFA, kinase insert domain receptor (KDR/VEGFR2) and plasmalemma vesicle-associated protein (PLVAP) gene expressions were detected during the long photoperiod. We also found that VEGFA and its receptor, VEGFR2, are expressed by neurons and tanycytes in both the ARH and ME. Altogether, these data show variations in the MBH vasculature according to seasons potentially through a VEGFA-dependent pathway, paving the way for future studies aiming to decipher the role of these changes in the hypothalamic control of seasonal reproduction.
ano.nymous@ccsd.cnrs.fr.invalid (Pierre-Marie Chevillard) 30 Apr 2024
https://hal.inrae.fr/hal-03746290v1
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[hal-03642441] Transcription of rRNA in early mouse embryos promotes chromatin reorganization and expression of major satellite repeats
During the first cell cycles of early development, the chromatin of the embryo is highly reprogrammed while the embryonic genome starts its own transcription. The spatial organization of the genome is an important process that contributes to regulating gene transcription in time and space. It has, however, been poorly studied in the context of early embryos. To study the cause-and-effect link between transcription and spatial organization in embryos, we focused on ribosomal genes, which are silent initially but start to be transcribed in 2-cell mouse embryos. We demonstrated that ribosomal sequences and early unprocessed rRNAs are spatially organized in a very particular manner between 2-cell and 16-cell stage. By using drugs that interfere with ribosomal DNA transcription, we showed that this organization – which is totally different in somatic cells – depends on an active transcription of ribosomal genes and induces a unique chromatin environment that favors transcription of major satellite sequences once the 4-cell stage has been reached.
ano.nymous@ccsd.cnrs.fr.invalid (Martine Chebrout) 15 Apr 2022
https://hal.science/hal-03642441v1
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[hal-03742076] The hepatokine FGF21 increases the human spermatozoa motility
Lifestyle, environment and excess body weight are not only associated with an increased risk of metabolic disorders, such as type 2 diabetes, but also to other pathological processes, such as infertility. A hormone produced mainly by the liver called fibroblast growth factor 21 (FGF21) is closely linked to the energy status and is increased in patients suffering from obesity or insulin resistance. Recently, FGF21 has been shown to be associated with female fertility disorders, but no or few data about the role of FGF21 on human male fertility has been described. In the present study, FGF21 was measured in the seminal fluid at a lower level in comparison to the blood level. Thus, in the present in vitro study, we aimed to decipher the FGF21 system in human semen. To evaluate the putative role of FGF21 on spermatozoa function, we incubated human spermatozoa with increasing concentrations of recombinant human FGF21. The FGF21 in seminal fluid is potentially produced by male reproductive tract tissues. In spermatozoa, the FGF21 signal was transduced by the two main receptors FGFR1-c and FGFR3 and the cofactor beta-klotho, which are colocalized in the middle piece of spermatozoa and stimulated the PI3K/Akt and MAPK pathways. Finally, in vitro treatment by FGF21 significantly increased sperm motility and ATP levels. Concomitantly, exposure to FGF21 improved the oxidative stress, as a lower ROS level was observed. Overall, these results seem to indicate that the metabolic factor, FGF21, positively modifies the activity and quality of the parameters of human spermatozoa.
ano.nymous@ccsd.cnrs.fr.invalid (Guillaume Bourdon) 02 Aug 2022
https://hal.inrae.fr/hal-03742076v1
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[hal-03555171] Prenatal maternal stress is associated with behavioural and epigenetic changes in Japanese quail
Prenatal maternal stress (PMS) influences many facets of offspring’s phenotype including morphology, behaviour and cognitive abilities. Recent research suggested that PMS also induced epigenetic modifications. In the present study, we analysed, in the Japanese quail, the effects of PMS on the emotional reactivity and cognitive abilities of the F1 offspring. We also investigated in the hippocampus, the paraventricular hypothalamic nucleus and subnuclei of the arcopallium/amygdala the level of two histone post-translational modifications, H3K4me2 and H3K27me3, known to be impacted by stress. We found that PMS does not affect F1 quail’s learning abilities but increases their emotional reactivity. Moreover, we demonstrated that PMS induced an increased density of H3K27me3 positive cells, in the hippocampus, paraventricular hypothalamic nucleus and dorsal nucleus of the amygdala, but not variations of H3K4me2. As these brain regions are involved in the control of vertebrates’ emotional responses, the effect of PMS on the epigenetic mark H3K27me3 could possibly be a mechanism involved in the behavioural effects we observed in F1 quail.
ano.nymous@ccsd.cnrs.fr.invalid (Marion Charrier) 30 Mar 2022
https://hal.science/hal-03555171v1
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[hal-03754006] How does apelin affect LH levels? An investigation at the level of GnRH and KNDy neurons
Hypothalamic control of reproduction relies on GnRH and kisspeptin (KP) secretions. KP neurons are sensitive to sex steroids and metabolic status and their distribution overlaps with neurons producing apelin, a metabolic hormone known to decrease LH secretion in rats. Here, we observed neuroanatomical contacts between apelin fibers and both KP and GnRH neurons in the hypothalamus of male rodents. Intracerebroventricular apelin infusion for 2 weeks in male mice did not decrease LH levels nor did it affect gene expression for KP, neurokinin B and dynorphin. Finally, increasing apelin concentrations did not modulate Ca2+ levels of cultured GnRH neurons, while 10 mu M apelin infusion on forskolin pretreated GnRH neurons revoked a rhythmic activity in 18% of GnRH neurons. These results suggest that acute apelin effect on LH secretion does not involve modulation of gene expression in KP neurons but may affect the secretory activity of GnRH neurons.
ano.nymous@ccsd.cnrs.fr.invalid (Anne Abot) 27 Oct 2022
https://hal.science/hal-03754006v1
