Kiss1 expression in the hypothalamic arcuate nucleus is lower in dairy cows of reduced fertility

We tested the hypothesis that divergent genetic merit for fertility of dairy cows is due to aberrant reproductive neuroendocrine function. The kisspeptin status of non-pregnant cows of either positive (POS) or negative (NEG) breeding values for fertility was studied in 3 groups (n = 8), based on their previous post-partum period: POS cows which had spontaneous ovarian cycles (POS-CYC) and NEG cows which either cycled (NEG-CYC) or did not cycle (NEG-NONCYC). Ovarian cycles were synchronized, blood samples were taken to define endocrine status and the animals were slaughtered in an artificial follicular phase. The brains and the pituitary glands were collected for quantitative polymerase chain reaction (qPCR) and in situ hybridisation (ISH) of hypothalamic GNRH1, Kiss1, TAC3 and PDYN and pituitary expression of LHB and FSHB. Gonadotropin releasing hormone (GnRH) and kisspeptin levels were quantified in snap frozen median eminentes (ME). GNRH1 expression and GnRH levels in the ME were similar across groups. Kiss1 expression in the preoptic area of the hypothalamus was also similar across groups, but Kiss1 in the arcuate nucleus was almost 2-fold higher in POS-CYC cows than in NEG groups. TAC3 expression was higher in POS-CYC cows. The number of pituitary gonadotropes and the level of expression of LHB and FSHB was similar across groups. We conclude that the lower levels of Kiss1 and TAC3 in NEG cows with low fertility status, and may lead to deficient GnRH and gonadotropin secretion.

The Role of TRPA1 Channels in the Central Processing of Odours Contributing to the Behavioural Responses of Mice

Transient receptor potential ankyrin 1 (TRPA1), a nonselective cation channel, contributes to several (patho)physiological processes. Smell loss is an early sign in several neurodegenerative disorders, such as multiple sclerosis, Parkinson’s and Alzheimer’s diseases; therefore, we focused on its role in olfaction and social behaviour with the aim to reveal its potential therapeutic use. The presence of Trpa1 mRNA was studied along the olfactory tract of mice by combined RNAscope in situ hybridisation and immunohistochemistry. The aversive effects of fox and cat odour were examined in parallel with stress hormone levels. In vitro calcium imaging was applied to test if these substances can directly activate TRPA1 receptors. The role of TRPA1 in social behaviour was investigated by comparing Trpa1 wild-type and knockout mice (KO). Trpa1 mRNA was detected in the olfactory bulb and piriform cortex, while its expression was weak in the olfactory epithelium. Fox, but not cat odour directly activated TRPA1 channels in TRPA1-overexpressing Chinese Hamster Ovary cell lines. Accordingly, KO animals showed less aversion against fox, but not cat odour. The social interest of KO mice was reduced during social habituation–dishabituation and social interaction, but not during resident–intruder tests. TRPA1 may contribute to odour processing at several points of the olfactory tract and may play an important role in shaping the social behaviour of mice. Thus, TRPA1 may influence the development of certain social disorders, serving as a potential drug target in the future.

An In Vitro Approach to Studying the Microbial Community and Impact of Pre and Probiotics under Anorexia Nervosa Related Dietary Restrictions

Individuals with anorexia nervosa (AN) often suffer psychological and gastrointestinal problems consistent with a dysregulated gut microbial community. Psychobiotics have been postulated to modify microbiota and improve mental well-being and gut symptoms, but there is currently a lack of evidence for such approaches in AN. The aim of this study was to use an in vitro colonic model to evaluate the impact of dietary restrictions associated with AN on the intestinal ecosystem and to assess the impact of pre and probiotic intervention. Bacteriology was quantified using flow cytometry combined with fluorescence in situ hybridisation and metabolic end products (including neurotransmitters) by gas chromatography and liquid chromatography mass spectrometry Consistent with previous research, the nutritional changes significantly reduced total microbiota and metabolites compared with healthy conditions. Pre and probiotic supplementation on restricted conditions enhanced the microbial community and modulated metabolic activity to resemble that of a healthy diet. The model system indicates that nutritional changes associated with AN can impact the microbial community, and that these changes can, at least in part, be restored through the use of pre and probiotic interventions.

The correlations between gut microbiota of Muslim Thai lactating women and their dietary intake and gut microbiota of breastfed infants

Introduction: Foods and nutrients are essential not only for human health, but also for the balance of gut microbiota. This research aimed to correlate the gut microbiota of lactating women with their food/ nutrient intakes, as well as with their infants’ gut microbiota. Methods: A cross-sectional study was conducted in 27 pairs of mothers and their exclusively breastfed infants. For lactating women, the dietary assessment was conducted by 24-hour recall, and food groups were assessed following the Food and Agriculture Organization’s guidelines, while nutrient intake was analysed using INMUNCAL V3 programme. Gut microbiota of mothers and infants were measured in stool samples using fluorescent in situ hybridisation technique. Results: It was found that energy intake of mothers was only 66% of the recommended Thai Dietary Reference Intakes (DRIs). Most micronutrient and dietary fibre intakes were below the Thai DRIs. Vitamin A (VA)-rich fruits and vegetables food group correlated positively with Lactobacillus species (spp). The association between gut microbiota and nutrient intake of lactating women showed that total protein, phosphorus, and VA were positively correlated with Bifidobacterium spp.; while β-carotene and vitamin C were also positively correlated with Lactobacillus spp. In contrast, consumption of eggs and calcium correlated negatively with Clostridium spp./ Enterobacter spp. Bifidobacterium spp. and Lactobacillus spp. of lactating women and breastfed infants showed strong correlations. Conclusion: Food and nutrient intakes of lactating women were correlated with their Clostridium spp./Enterobacter spp., Bifidobacterium spp. and Lactobacillus spp. Furthermore, Bifidobacterium spp. and Lactobacillus spp. of mothers and breastfed infants showed strong correlations.

TELO-SCOPE study: a randomised, double-blind, placebo-controlled, phase 2 trial of danazol for short telomere related pulmonary fibrosis

IntroductionRecent discoveries have identified shortened telomeres and related mutations in people with pulmonary fibrosis (PF). There is evidence to suggest that androgens, including danazol, may be effective in lengthening telomeres in peripheral blood cells. This study aims to assess the safety and efficacy of danazol in adults and children with PF associated with telomere shortening.Methods and analysisA multi-centre, double-blind, placebo-controlled, randomised trial of danazol will be conducted in subjects aged >5 years with PF associated with age-adjusted telomere length ≤10th centile measured by flow fluorescence in situ hybridisation; or in children, a diagnosis of dyskeratosis congenita. Adult participants will receive danazol 800 mg daily in two divided doses or identical placebo capsules orally for 12 months, in addition to standard of care (including pirfenidone or nintedanib). Paediatric participants will receive danazol 2 mg/kg/day orally in two divided doses or identical placebo for 6 months. If no side effects are encountered, the dose will be escalated to 4 mg/kg/day (maximum 800 mg daily) orally in two divided doses for a further 6 months. The primary outcome is change in absolute telomere length in base pairs, measured using the telomere shortest length assay (TeSLA), at 12 months in the intention to treat population.Ethics and disseminationEthics approval has been granted in Australia by the Metro South Human Research Ethics Committee (HREC/2020/QMS/66385). The study will be conducted and reported according to Standard Protocol Items: Recommendations for Interventional Trials guidelines. Results will be published in peer-reviewed journals and presented at international and national conferences.Trial registration numbersNCT04638517; Australian New Zealand Clinical Trials Registry (ACTRN12620001363976p).

The intraovarian cellular origins of GDF9 and BMP15 in the mouse and aspects of their biological properties

<p>Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are both members of the TGF-ß protein superfamily and are known to be essential for normal follicular development in mammals. Several studies have highlighted the species-specific effects of BMP15 and GDF9, which could be attributed, at least in part to the differences in the follicular expression patterns and to different forms of the secreted proteins. In the mouse, GDF9 is required for follicular development, whereas BMP15 appears to be only required near ovulation with contradictory reports as to the timing of BMP15 expression. However, mouse BMP15 and GDF9 are known to have the capability of acting together synergistically. The aims of this thesis were to characterise in the mouse ovary, the expression patterns (localisation and levels) of Bmp15 and Gdf9 mRNA throughout follicular development, and to determine the peri-ovulatory expression of the corresponding proteins. In situ hybridisation and quantitative PCR analyses of ovarian samples and follicular cells collected from control and superovulated mice confirmed that Gdf9 and Bmp15 mRNA are expressed exclusively in oocytes from primary and early secondary stage follicles respectively. qPCR analysis of denuded oocytes (DO) revealed a tight correlation, and therefore co-regulation, between the expression levels of Bmp15 and Gdf9 irrespective of follicular developmental stage, with steady expression until the preovulatory LH surge when down-regulation of Bmp15 and Gdf9 occurred. Throughout the follicular developmental stages examined, Gdf9 was expressed in greater abundance relative to Bmp15, with a Bmp15:Gdf9 mRNA ratio of 1:4.12. [...] In conclusion, oocyte-derived Bmp15 and Gdf9 mRNA expression is co-regulated throughout follicular development in mice, with Gdf9 being more abundant than Bmp15, which might be an important factor in determining high ovulation quota. The expression of the target genes is down-regulated as the oocyte reaches developmental competence following the preovulatory LH surge. Protein expression data provided evidence that in vivo the immature mouse oocyte is capable of secreting all BMP15 protein forms previously detected in vitro. After the preovulatory LH surge, all visible protein forms are associated with the somatic follicular cells, in particular with the expanded cumulus mass. Of particular interest is the presence of the large protein complexes in the cumulus cell lysates, which suggests a storage and activation process involving ECM proteins, similar to the mechanism reported for other TGF-ß superfamily members, such as TGF-ß1 and myostatin. The finding that the BMP15 precursor protein is biologically active with a different activity to that of the processed mature protein form suggests that the full-length precursor protein may regulate or provide at least a portion of the biological activity of BMP15 in mice.</p>

The intraovarian cellular origins of GDF9 and BMP15 in the mouse and aspects of their biological properties

<p>Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are both members of the TGF-ß protein superfamily and are known to be essential for normal follicular development in mammals. Several studies have highlighted the species-specific effects of BMP15 and GDF9, which could be attributed, at least in part to the differences in the follicular expression patterns and to different forms of the secreted proteins. In the mouse, GDF9 is required for follicular development, whereas BMP15 appears to be only required near ovulation with contradictory reports as to the timing of BMP15 expression. However, mouse BMP15 and GDF9 are known to have the capability of acting together synergistically. The aims of this thesis were to characterise in the mouse ovary, the expression patterns (localisation and levels) of Bmp15 and Gdf9 mRNA throughout follicular development, and to determine the peri-ovulatory expression of the corresponding proteins. In situ hybridisation and quantitative PCR analyses of ovarian samples and follicular cells collected from control and superovulated mice confirmed that Gdf9 and Bmp15 mRNA are expressed exclusively in oocytes from primary and early secondary stage follicles respectively. qPCR analysis of denuded oocytes (DO) revealed a tight correlation, and therefore co-regulation, between the expression levels of Bmp15 and Gdf9 irrespective of follicular developmental stage, with steady expression until the preovulatory LH surge when down-regulation of Bmp15 and Gdf9 occurred. Throughout the follicular developmental stages examined, Gdf9 was expressed in greater abundance relative to Bmp15, with a Bmp15:Gdf9 mRNA ratio of 1:4.12. [...] In conclusion, oocyte-derived Bmp15 and Gdf9 mRNA expression is co-regulated throughout follicular development in mice, with Gdf9 being more abundant than Bmp15, which might be an important factor in determining high ovulation quota. The expression of the target genes is down-regulated as the oocyte reaches developmental competence following the preovulatory LH surge. Protein expression data provided evidence that in vivo the immature mouse oocyte is capable of secreting all BMP15 protein forms previously detected in vitro. After the preovulatory LH surge, all visible protein forms are associated with the somatic follicular cells, in particular with the expanded cumulus mass. Of particular interest is the presence of the large protein complexes in the cumulus cell lysates, which suggests a storage and activation process involving ECM proteins, similar to the mechanism reported for other TGF-ß superfamily members, such as TGF-ß1 and myostatin. The finding that the BMP15 precursor protein is biologically active with a different activity to that of the processed mature protein form suggests that the full-length precursor protein may regulate or provide at least a portion of the biological activity of BMP15 in mice.</p>

(Un)expected Similarity of the Temporary Adhesive Systems of Marine, Brackish, and Freshwater Flatworms

Many free-living flatworms have evolved a temporary adhesion system, which allows them to quickly attach to and release from diverse substrates. In the marine Macrostomum lignano, the morphology of the adhesive system and the adhesion-related proteins have been characterised. However, little is known about how temporary adhesion is performed in other aquatic environments. Here, we performed a 3D reconstruction of the M. lignano adhesive organ and compared it to the morphology of five selected Macrostomum, representing two marine, one brackish, and two freshwater species. We compared the protein domains of the two adhesive proteins, as well as an anchor cell-specific intermediate filament. We analysed the gene expression of these proteins by in situ hybridisation and performed functional knockdowns with RNA interference. Remarkably, there are almost no differences in terms of morphology, protein regions, and gene expression based on marine, brackish, and freshwater habitats. This implies that glue components produced by macrostomids are conserved among species, and this set of two-component glue functions from low to high salinity. These findings could contribute to the development of novel reversible biomimetic glues that work in all wet environments and could have applications in drug delivery systems, tissue adhesives, or wound dressings.