Prenatal Androgen Exposure Induces Anxiety-Like Behavior in Ewes.

INTRODUCTION: In humans, prenatal androgen excess can lead to a broad spectrum of pathologies in adulthood, including polycystic ovary syndrome (PCOS). Women with PCOS present a variety of reproductive and metabolic disturbances and they also face increased risk to develop neuropsychiatric disorders such as depression and anxiety. Despite the high prevalence, the cause of depressive and anxiety symptoms is not fully elucidated. The use of androgenized ewe models can provide valuable insights into the pathogenesis of PCOS, as they closely mimic the reproductive, neuroendocrine, and metabolic characteristics observed in women with this condition. METHOD: We studied the impact of prenatal exposure to testosterone propionate on cognitive and behavioral performances of Ile-de-France ewes, using a plethora of behavioral tests for anxiety and cognitive performances. RESULTS: Our findings indicate that prenatal androgenized ewes exhibit markedly elevated levels of anxiety-like behavior compared to control animals, while showing no discernible differences in cognitive performance. CONCLUSION: These discoveries offer novel perspectives on how maternal androgen excess contributes to anxiogenic effects in PCOS preclinical models, underscoring the ewe's significance as a model for conducting mechanistic studies to unravel the physiological and molecular aspects of anxiety.

Quantitation of endogenous GnRH by validated nano-HPLC-HRMS method: a pilot study on ewe plasma.

Gonadotropin-releasing hormone isoform I (GnRH), a neuro-deca-peptide, plays a fundamental role in development and maintenance of the reproductive system in vertebrates. The anomalous release of GnRH is observed in reproductive disorder such as hypogonadotropic hypogonadism, polycystic ovary syndrome (PCOS), or following prenatal exposure to elevated androgen levels. Quantitation of GnRH plasma levels could help to diagnose and better understand these pathologies. Here, a validated nano-high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS) method to quantify GnRH in ewe plasma samples is presented. Protein precipitation and solid-phase extraction (SPE) pre-treatment steps were required to purify and enrich GnRH and internal standard (lamprey-luteinizing hormone-releasing hormone-III, l-LHRH-III). For the validation process, a surrogate matrix approach was chosen following the International Council for Harmonisation (ICH) and FDA guidelines. Before the validation study, the validation model using the surrogate matrix was compared with those using a real matrix such as human plasma. All the tested parameters were analogous confirming the use of the surrogate matrix as a standard calibration medium. From the validation study, limit of detection (LOD) and limit of quantitation (LOQ) values of 0.008 and 0.024 ng/mL were obtained, respectively. Selectivity, accuracy, precision, recovery, and matrix effect were assessed with quality control samples in human plasma and all values were acceptable. Sixteen samples belonging to healthy and prenatal androgen (PNA) exposed ewes were collected and analyzed, and the GnRH levels ranged between 0.05 and 3.26 ng/mL. The nano-HPLC-HRMS developed here was successful in measuring GnRH, representing therefore a suitable technique to quantify GnRH in ewe plasma and to detect it in other matrices and species.

The olfactory secretome varies according to season in female sheep and goat.

BACKGROUND: Small ungulates (sheep and goat) display a seasonal breeding, characterised by two successive periods, sexual activity (SA) and sexual rest (SR). Odours emitted by a sexually active male can reactivate the ovulatory cycle of anoestrus females. The plasticity of the olfactory system under these hormonal changes has never been explored at the peripheral level of odours reception. As it was shown in pig that the olfactory secretome (proteins secreted in the nasal mucus) could be modified under hormonal control, we monitored its composition in females of both species through several reproductive seasons, thanks to a non-invasive sampling of olfactory mucus. For this purpose, two-dimensional gel electrophoresis (2D-E), western-blot with specific antibodies, MALDI-TOF and high-resolution (nano-LC-MS/MS) mass spectrometry, RACE-PCR and molecular modelling were used. RESULTS: In both species the olfactory secretome is composed of isoforms of OBP-like proteins, generated by post-translational modifications, as phosphorylation, N-glycosylation and O-GlcNAcylation. Important changes were observed in the olfactory secretome between the sexual rest and the sexual activity periods, characterised in ewe by the specific expression of SAL-like proteins and the emergence of OBPs O-GlcNAcylation. In goat, the differences between SA and SR did not come from new proteins expression, but from different post-translational modifications, the main difference between the SA and SR secretome being the number of isoforms of each protein. Proteomics data are available via ProteomeXchange with identifier PXD014833. CONCLUSION: Despite common behaviour, seasonal breeding, and genetic resources, the two species seem to adapt their olfactory equipment in SA by different modalities: the variation of olfactory secretome in ewe could correspond to a specialization to detect male odours only in SA, whereas in goat the stability of the olfactory secretome could indicate a constant capacity of odours detection suggesting that the hallmark of SA in goat might be the emission of specific odours by the sexually active male. In both species, the olfactory secretome is a phenotype reflecting the physiological status of females, and could be used by breeders to monitor their receptivity to the male effect.

Sexually active bucks are a critical social cue that activates the gonadotrope axis and early puberty onset in does.

In rodents, early exposure to adult male is well known to induce an early puberty in females (Vandenbergh effect). This phenomenon has been less studied in other mammals. In goats, despite our extensive knowledge about the "male-effect" phenomenon in adults (i.e. ovulation induced by the introduction of the male during the anestrous), there are few data on the consequences of an early exposure of females to males. Here, we evaluated the puberty onset of young alpine goats when raised since weaning with intact bucks (INT), with castrated bucks (CAS) or isolated from bucks (ISOL). The INT group had the first ovulation 1.5 month before the two other groups. Despite the earlier puberty the INT group of females had normal and regular ovarian cycles. Morphological study of the genital tract showed that at 6 months, uterus of INT goats was 40% heavier than CAS and ISOL goats. Moreover, INT females had a myometrium significantly thicker and INT was the only group having corpora lutea. In our study, INT females were pubescent in the month following the entry of bucks into the breeding season, suggesting that only sexually active bucks provide the signal responsible for puberty acceleration. By removing direct contact with the bucks, we showed that somatosensory interactions were dispensable for an early puberty induction. Finally, no difference in the GnRH network (fiber density and number of synaptic appositions) can be detected between pubescent and non-pubescent females, suggesting that the male stimulations triggering puberty onset act probably on upstream neuronal networks, potentially on kisspeptin neurons.

Cutaneous application of SecurePig® FLASH, a Pig appeasing pheromone analogue, facilitates adaptation and manages social behavior during feeding in semi-extensive conditions.

BACKGROUND: Farm animals face several challenges throughout their lives, which can affect both their welfare and their productivity. Promoting adaptation in animals is one way of limiting these consequences. In various animal species, the use of maternal appeasing pheromones is efficient to reduce aggressiveness, improve adaptation and thus ensuring better welfare and productivity. This study sought to investigate the efficiency of a treatment with a Pig Appeasing Pheromone (PAP) on the behavior of pigs reared under semi-extensive conditions and exposed to a potential conflict- collective feeding. Animals (n = 14 divided in 2 groups of 7) were subjected to 3 different phases, (A) baseline - no pigs received the PAP, (B) SP - 2 out of the 7 pigs per group received the PAP and (C) AP- all pigs received the PAP. Behaviors related to feeding, aggression and locomotion were compared between the 3 phases of the study. RESULTS: Compared to the baseline period, we observed that the number of head knocks was reduced when some pigs (p < 0.001) and all pigs (p < 0.005) received the PAP. Similarly, we observed that the number of fleeing attempts was reduced when some pigs (p < 0.001) and all pigs (p < 0.001) were treated when compared to baseline. This number was lower in the AP phase than in the SP phase (p < 0.001). When all pigs were treated (AP), we also observed that they spent less time investigating the floor than during the two other phases (p < 0.001), but they seemed more likely to leave the feeder due to the presence or behavior of another pig of the group (SP vs. AP, p < 0.05). CONCLUSIONS: The PAP application improved adaptation in pigs by reducing aggressiveness and promoting conflict avoidance. Those results validate the efficiency of the pheromonal treatment under semi-extensive rearing conditions to help pigs to cope with a challenging situation. Using PAP in the pig industry seems interesting to limit unwanted consequences of farm practices on animal welfare and productivity, by promoting their adaptation.

Horses Solve Visible but Not Invisible Displacement Tasks in an Object Permanence Paradigm.

A key question in the field of animal cognition is how animals comprehend their physical world. Object permanence is one of the fundamental features of physical cognition. It is the ability to reason about hidden objects and to mentally reconstruct their invisible displacements. This cognitive skill has been studied in a wide range of species but never directly in the horse (Equus caballus). In this study, we therefore assessed the understanding of visible and invisible displacements in adult Welsh mares in two complementary experiments, using different horses. In experiment 1, visible displacement was investigated using two tasks adapted from the Uzgiris and Hunt scale 1. Invisible displacement was assessed using a transposition task, in which food was first hidden in one of two containers and the location of the containers was then switched. In experiment 2, we further investigated horses' understanding of visible and invisible displacements using an easier procedure designed to avoid potentially confounding factors. In both experiments, horses successfully completed the tasks involving visible displacement with two or three possible hiding places. However, in both experiments, horses failed the transposition tasks, suggesting that they may not be able to track the displacement of an object that is not directly perceived (i.e., invisible displacement). These results bring new insights into object permanence in horses and how they represent their physical world.

The Preoptic Area and the RFamide-Related Peptide Neuronal System Gate Seasonal Changes in Chemosensory Processing.

Males of many species rely on chemosensory information for social communication. In male Syrian hamsters (Mesocricetus auratus), as in many species, female chemosignals potently stimulate sexual behavior and a concurrent, rapid increase in circulating luteinizing hormone (LH) and testosterone (T). However, under winter-like, short-day (SD) photoperiods, when Syrian hamsters are reproductively quiescent, these same female chemosignals fail to elicit behavioral or hormonal responses, even after T replacement. It is currently unknown where in the brain chemosensory processing is gated in a seasonally dependent manner such that reproductive responses are only displayed during the appropriate breeding season. The goal of the present study was to determine where this gating occurred by identifying neural loci that respond differentially to female chemosignals across photoperiods, independent of circulating T concentrations. Adult male Syrian hamsters were housed under either long-day (LD) (reproductively active) or SD (reproductively inactive) photoperiods with half of the SD animals receiving T replacement. Animals were exposed to either female hamster vaginal secretions (FHVSs) diluted in mineral oil or to vehicle, and the activational state of chemosensory processing centers and elements of the neuroendocrine reproductive axis were examined. Components of the chemosensory pathway upstream of hypothalamic centers increased expression of FOS, an indirect marker of neuronal activation, similarly across photoperiods. In contrast, the preoptic area (POA) of the hypothalamus responded to FHVS only in LD animals, consistent with its role in promoting expression of male sexual behavior. Within the neuroendocrine axis, the RF-amide related peptide (RFRP), but not the kisspeptin neuronal system responded to FHVS only in LD animals. Neither response within the POA or the RFRP neuronal system was rescued by T replacement in SD animals, mirroring photoperiodic regulation of reproductive responses. Considering the POA and the RFRP neuronal system promote reproductive behavior and function in male Syrian hamsters, differential activation of these systems represents a potential means by which photoperiod limits expression of reproduction to the appropriate environmental context.

Sexually active bucks are efficient to stimulate female ovulatory activity during the anestrous season also under temperate latitudes.

Goats are seasonal breeders and photoperiod is the main cue controlling the onset and offset of the breeding season. Nevertheless introducing a sexually active buck in a group of females during anestrous can stimulate their reproductive function and induce ovulation. This "male-effect" is very efficient under subtropical latitudes, when using sexually active males previously stimulated by a photoperiodic treatment. However, there is less evidence of its feasibility under temperate latitudes where the more important variation in day length could be responsible for a stronger inhibition of female sexual activity. The aim of this study was therefore to determine whether intense sexual activity can be induced in alpine bucks during the non-breeding season by a long-day treatment under temperate latitude and if these males could be used to produce an efficient male-effect. Bucks (n=21) were divided in two groups, one submitted to a photoperiodic treatment from November 1st to January 15th and then switched to natural photoperiod, while the other group remained entirely under the natural photoperiod. The ones submitted to this light treatment exhibit higher testicular volume and testosterone level 6 weeks after the end of the treatment. At the end of March, bucks were used to stimulate anestrous does (n=41) continuously for 15 days. We showed that (a) light treatment was efficient to induce an increase of sexual activity in bucks and (b) that the introduction of stimulated bucks among females induced a significantly higher proportion of ovulation in anestrous does than control bucks (86% vs 5%). Our results indicate that under temperate latitudes induction of ovulation in females during the anestrous season is feasible using bucks treated with long-days during winter.

Tribbles expression in cumulus cells is related to oocyte maturation and fatty acid metabolism.

BACKGROUND: In mammals, the Tribbles family includes widely expressed serine-threonine kinase-like proteins (TRIB1, TRIB2 and TRIB3) that are involved in multiple biological processes including cell proliferation and fatty acid (FA) metabolism. Our recent studies highlighted the importance of FA metabolism in cumulus cells (CC) during oocyte maturation in vertebrates and reported a higher TRIB1 expression in CC surrounding in vivo mature oocytes as compared to immature ooocytes in mice and cows. The objective was to investigate Tribbles expression patterns in bovine CC during in vitro maturation (IVM) and to examine their roles in the cumulus-oocyte complex. METHODS: Tribbles gene expression was analyzed in bovine and murine CC using quantitative RT-PCR. Proteins were detected using Western blot and intracellular localization was assessed by immunofluorescence. Bovine COCs were treated with etomoxir, an inhibitor of FA oxidation (FAO) which blocks CPT1 activity, during 6 h and 18 h IVM. Oocyte meiotic stage was assessed and expression of Tribbles and lipid metabolism genes was quantified in CC. RESULTS AND DISCUSSION: TRIB1 and TRIB3 were more strongly expressed whereas TRIB2 was less expressed in CC surrounding the oocytes from preovulatory follicles than in CC of immature ones. In CC, Tribbles were located in the cytoplasm and nucleus; in mitotic cells TRIB2 and TRIB3 were detected in the spindle. In the oocyte, Tribbles proteins were detected in the ooplasm; also TRIB2 and TRIB3 were more accumulated in the germinal vesicle. In bovine CC, expression of TRIB1 and TRIB3 was transiently increased at a time preceding oocyte meiosis resumption in vitro. Treatment with etomoxir (150 µM) during IVM resulted in a significant reduction of oocyte maturation rate and decreased MAPK3/1 phosphorylation in the oocytes. In CC, 18 h IVM of etomoxir treatment significantly increased expression of TRIB1, TRIB3, CPTA1 (enzyme regulating FA entry in mitochondria for FAO) and CD36 (thrombospondin receptor involved in FA transport). Under the same conditions, expression of TRIB2 and ACACA (Acetyl coenzyme A carboxylase involved in FA synthesis) decreased in CC. All considered, Tribbles family members may be involved in cell proliferation and in FAO signaling in CC and participate in oocyte meiotic resumption regulation.