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Microsatellite identification of ramet genotypes in a clonal plant Iris pumila
No full textIris pumila L. is a perennial species that reproduces sexually and asexually by underground rhizomes (clonal growth). Clonal plants form spatial networks of ramets that may include: a) groupings of individuals of different genetics that have arisen from seeds, i.e. by sexual reproduction; b) groupings of individuals that originate from the clonal growth of a single genet; or c) groupings of individuals that have arisen from both sexual reproduction and clonal growth. For this reason, when sampling individuals in the field, it is difficult to determine with certainty whether the sampled plants belong to the same or different genotypes. I. pumila also exhibits significant genetic polymorphism in flower colour, which could be used to identify genetically distinct clones in the wild.
Since plant mating occurs more frequently among neighboring individuals, the question of whether clonality affects mating should depend on how clonal growth affects the spatial relationships between ramets of different genotypes. The plants of I. pumila follow the “phalanx strategy“ of clonality and form a compact structure of closely spaced ramets. The close grouping of ramets around the parental shoot could potentially limit the intermixing of ramets from different clones. These restrictions on the mixing of clones could have important implications for mating and fertility.
Currently, several techniques using molecular markers allow the unambiguous definition of individual genes and have been used with great success for clonal plants. Highly polymorphic genetic markers offer excellent opportunity to gain new insights into the functional interactions between sexual and clonal reproduction in plants.
The original premise of experimental research was that it is possible to distinguish plant clones by analysing microsatellite loci, i.e. by detecting the so-called "multilocus allele phenotypes". If a difference between individuals is detected at any locus of a microsatellite marker, we can conclude that the individuals are genetically different and do not represent the same clone. We have assumed that groups of ramets that are very close to each other mostly represent parts of the same clone (genetically identical ramets), while more distant ramets could be parts of different genotypes, i.e. clones.
As no information, to the best of our knowledge, on the use of molecular microsatellite markers in the study of I. pumila could be found in the literature, data on the expression of microsatellite markers (EST-SSR) in transcriptomes of other Iris species were used. The selection of EST-SSR was based on whether the markers were expressed in closely related species and whether they showed polymorphism. The final analysis was performed with 5 microsatellite loci. In the overall analysis, more than 40 alleles were detected, of which only one is monomorphic and non-informative. The polymorphism levels of all markers generally proved to be high and sufficient to distinguish the individuals, with the exception of a single marker whose amplification revealed only four different alleles.Abstract Book: Congress of the European Sociey for Evolutionary Biology; 2025 Aug 17-22; Barcelona, Spain. European Society for Evolutionary Biology; 2025. p. 1611
Norepinephrine-induced intracellular Ca2+ increase is coupled with astrocytic BK channel activation and capillary response
No full textAstrocytes are abundant glial cells organized in a meshwork in which each cell is in contact with both neuronal and vascular elements. They receive and respond to neuronal signals and modulate synaptic activity and diameter of blood vessels through changes in their intracellular Ca2+. Norepinephrine plays an important role in both of these astrocytic functions; however, it remains unclear whether norepinephrine-induced intracellular Ca2+ increase leads to further cellular adjustments in astrocyte
activity. Here, we reveal a causal relationship between norepinephrine-induced intracellular Ca2+ increase, α1-adrenergic receptor activation and activation of large-conductance Ca2+ -dependent potassium ion (BK) channel in cultured rat cortical astrocytes. BK channel activation was abolished by α1-adrenergic receptor blockade, depletion of intracellular Ca2+ stores, or dialysis of astrocytes with a Ca2+ chelator. We further show that this norepinephrine-induced astrocytic Ca2+ -BK channel coupling contributes to a reduction in cortical capillary diameter. The capillary response was prevented by pharmacological silencing of astrocytes or BK channel blockade, whereas the norepinephrine effect was mimicked by direct BK channel activation with an agonist in acute brain slices. In summary, these results elucidate a previously unrecognized cellular response of astrocytes to norepinephrine that is coupled with modulation of capillary diameter and may represent an integral part of the astrocytic communication with neurons and blood vessels
Long-term dietary restriction changes lipid homeostasis and consequently impairs testosterone production in aged Wistar rats
No full textTestosterone (T) is a central androgen responsible for the maintenance of vegetative
and reproductive functions and sexual behavior in males. T is mainly synthesized
through the process of testicular steroidogenesis with cholesterol (CHOL) as the
initial precursor. It is known that T levels gradually decrease, along with an increase
in CHOL, LDL-C, triglycerides (TG), and a decrease in HDL-C in advanced stages
of life. Dietary restriction (DR) ameliorates lipid status and raises T levels in obese
and overweight men. Here, we investigated whether the beneficial effects of DR
on serum lipid status consequently improve T production at advanced stages;
therefore, we exposed male Wistar rats to long-term DR (LTDR, 18 months).
We confirmed an age-related decrease in serum T levels, reduced expression
of genes and proteins of steroidogenic machinery with a simultaneous increase
in serum CHOL, LDL-C, and TG levels. LTDR additionally decreased T synthesis,
expression of Star/StAR and Cyp11a1/CYP11A1, and testicular CHOL levels. At the
same time, LTDR reduced serum CHOL, LDL-C, and TG levels and increased HDL-C
levels. To confirm that the effects of DR are determined by the duration of the
treatment, we also checked the effects of the short-term DR (STDR, 3 months)
and demonstrated that STDR did not alter T levels and did not affect serum lipids.
Our results indicate the importance of sustained systemic lipid homeostasis for
T production in advanced life stages and show that the effects of restricted food
intake on testicular androgen capacity depend on the duration of DR
Validation of suitable reference genes for qPCR expression analysis in young developing in vitro grown kohlarabi plants during different hormone and sugar stimuli
No full textReference genes (RGs) are used as internal controls to normalize gene expression data in quantitative real-time polymerase chain reaction (qPCR) analysis. It is assumed that these genes exhibit consistent expression levels across various experimental conditions, tissues, and developmental stages since they have critical functions in plant cells and metabolism. However, it was shown that there is a need for systematical assessment of their stability under various conditions in order to ensure more accurate and reliable results. Previously, our research group developed a competent in vitro model system for kohlrabi de novo shoot organogenesis that includes four early developmental stages of kohlrabi plants under varying hormone and sugar treatments. Since stability of potential RGs has not yet been investigated in kohlrabi it was crucial to validate their constancy for our own specific experimental setup. For validation of 15 selected RGs we used online software RefFinder that integrates four different statistical algorithms (geNorm, NormFinder, BestKeeper and DCq method) by calculating geometric mean of these approaches, determining the final ranking list. Comprehensive ranking showed that ACT7 and TUA2 along with SAND, PP2A, eIF4A-1 and H3.3 were the most stable RGs throughout the experimental subsets, while GAPB and GLUR3.2 were the lowest ranked ones. This was followed by qPCR validation using relevant sucrose-related target genes. Overall, we confirmed necessity in optimization of gene expression data normalization and consider our study a very helpful instrument in terms of choosing appropriate reference genes in plants that are not used as common model systems, such as kohlrabi.Golub D, editor. Book of asbtracts: V Symposium of biologists and ecologists of Republic of Srpska SBERS2025; 2025 Nov 13-25; Banja Luka, Bosnia and Herzegovina. Banja Luka: Faculty of Natural Sciences and Mathematics, University of Banja Luka; 2025. p. 34
Common and sex-specific roles of protein tyrosine phosphatase receptors N and N2 in the regulation of the (neuro)endocrine system
No full textThe protein tyrosine phosphatase receptors N and N2 are encoded by the Ptprn
and Ptprn2 genes, which are expressed in neuroendocrine cells of the
hypothalamus, pituitary gland, and diffuse neuroendocrine system, including the
pancreas, lung, and intestine. PTPRN and PTPRN2 lack protein tyrosine
phosphatase activity due to the mutation of two residues in their intracellular
catalytic domains. Here, we analyzed the roles of PTPRN and PTPRN2 in the
regulation of the neuroendocrine system. Regarding functionality of the
hypothalamic-pituitary-gonadal axis, simultaneous knockout of the Ptprn and
Ptprn2 causes infertility in female mice, while males are fertile. DKO females
displayed delayed puberty, lack of ovulation, and changes in ovarian gene
expression and steroidogenesis. DKO females and males showed decreased
gonadotropins synthesis in the pituitary. Based on the analysis of hypothalamic
functionality, our data indicate a critical role of Ptprn and Ptprn2 in kisspeptin -
gonadotropin-realising (GnRH) hormone neuronal function and sexual dimorphism
in the threshold levels of GnRH required to preserve reproductive functions.
Related to the hypothalamic-pituitary-adrenal axis, hyperplasia of the pituitary
intermediate lobe, an increase in the expression of the corticotroph/melanotroph
genes (Pomc and Tbx19), and the melanotroph-specific gene (Pax7) were
detected in DKO females and males. Synthesis and release of beta-endorphin,
alpha-melanocyte-stimulating hormone, adrenocorticotropic hormone, serum
corticosterone, and adrenal mass were elevated in DKO mice. These results
indicate that PTPRNs contribute to hypothalamic-pituitary-adrenal function by
being involved in processes governing postnatal melanotroph development
regardless of sex. Regardless of sex specificity, DKO also affected the synthesis
of oxytocin, growth hormone, and thyroid stimulating hormone subunit beta.Dragić M, editor. Book of abstracts: 9th Congress of Serbian Neuroscience Society; 2025 Oct 6-8; Belgrade, Serbia. Belgrade: Serbian Neuroscience Society; 2025
Effects of liraglutide and exercise on liver function in middle-aged rats
No full textHepatic function progressively declines with age, contributing to metabolic dysregulation and increased susceptibility to chronic diseases. The liver plays a central role in metabolism regulation, detoxification, and systemic homeostasis, thus, preserving its function is essential for healthy aging. While liraglutide and strength- endurance training have demonstrated efficacy in managing type 2 diabetes and obesity, their potential benefits in healthy middle-aged individuals remains poorly understood. To address this, we first compared young (CY; 4-month-old) and middle-aged (C; 16- month-old) male rats to assess age-related changes. Next, middle-aged rats were treated with liraglutide (L; Saxenda® 0.186 mg/kg/day subcutaneously, human equivalent dose 1.8 mg), subjected to strength-endurance training (E; ladder climbing 3x/week with 25–50% body weight load), or both (L+E), to evaluate effects on liver morphology and function. Middle-aged males showed lower (p<0.05) serum testosterone, increased (p<0.05) adipose tissue-to-body weight ratio, decreased (p<0.05) liver-to-body weight ratio, and elevated (p<0.05) serum cholesterol and non-HDL cholesterol, but preserved glycemic control compared to young rats. Liraglutide significantly reduced (p<0.001) body mass and visceral fat. Triglycerides serum concentration decreased (p<0.05) in L and L+E groups compared to C. Hepatic Hmgcr expression, encoding the rate-limiting enzyme in cholesterol biosynthesis, was downregulated (p<0.05) in the E group, while Cyp7a1, responsible for cholesterol catabolism, was significantly upregulated (p < 0.001) in L+E. Additionally, Fasn expression, essential for lipogenesis, decreased (p<0.05) in E and L+E groups. Hepatic ALT levels were reduced (p< 0.05) in L group, indicating hepatoprotection. Oxidative stress and lipid peroxidation indexes in the liver were elevated by exercise alone but were significantly lower (p<0.05) in both L and L+E groups compared to controls or exercise alone, demonstrating that liraglutide mitigated the exercise-induced redox imbalance. However, PAS staining revealed increased (p<0.05) glycogen content only in the E group. In summary, liraglutide improved metabolic health and liver function while reducing exercise-induced oxidative stress. When combined with exercise, it modulated the expression of hepatic genes involved in lipid metabolism. These findings suggest overall benefits for liver health in middle-aged rats.Meeting Abstract Book: 12th ESE Young Endocrinologists and Scientists (EYES); 2025 Sep 26-28; Milan, Italy. Oxford: University Press; 2025. p. i24-5. (European Journal of Endocrinology; Vol. 193; Suppl. 1)
Comparative Evaluation of Radical Scavenging Activity and Phytochemical Composition of Vitis vinifera L. cv. Vranac Grape Skin Extracts Obtained by Green Extraction Techniques
No full textThe valorization of winemaking by-products such as grape skins has gained increasing attention due to their richness in polyphenols with notable antioxidant properties. In this study, we aimed to investigate the radical scavenging potential and phytochemical composition of Vitis vinifera L. cv. Vranac grape skin extracts obtained using three environmentally friendly extraction techniques: supercritical CO₂ extraction (SCE), pressurized liquid extraction (PLE), and ultrasound -assisted extraction (UAE). Grape skins were separated from the Vranac pomace, freeze-dried, and subjected to three extraction protocols: (1) SCE with pure CO₂ at 40 °C and 200 bar for 90 minutes, (2) PLE under identical conditions using 50% ethanol as cosolvent in a 1:10 (w/v) solid-to-liquid ratio, and (3) UAE using 50% ethanol at the same solid-to-liquid ratio. Electron paramagnetic resonance (EPR) spectroscopy was used to quantify the extracts' ability to scavenge DPPH, hydroxyl (•OH), and superoxide (O₂•⁻) radicals. High-resolution mass spectrometry (Orbitrap-MS) was employed to analyze the composition of polyphenolic compounds in the extracts. EPR analysis demonstrated that all extracts exhibited free radical scavenging activity, with significant variation among methods. The SCE extract, prepared without a cosolvent, showed limited activity toward DPPH (15.62%) but high efficacy against •OH (69.14%) and O₂•⁻ (81.55%) radicals. The PLE extract showed the highest DPPH scavenging (37.86%) along with strong activity against •OH (80.86%) and O₂•⁻ (83.20%). Similarly, the UAE extract exhibited substantial antioxidant potential with DPPH (32.77%), •OH (83.06%), and O₂•⁻ (83.40%) scavenging capacities. Orbitrap-MS analysis revealed a broad range of flavonoids, flavonols, and anthocyanins in both ethanol-based extracts, particularly PLE, which contained high levels of quercetin, malvidin derivatives, myricetin glycosides, and other bioactive phenolics. UAE also recovered a diverse spectrum of antioxidant compounds, albeit at slightly lower levels. In contrast, the SCE extract contained only trace amounts of a few low-polarity flavonoids, consistent with the absence of a cosolvent and the extract's reduced DPPH scavenging activity. The extraction method plays a key role in shaping the antioxidant activity and polyphenolic content of Vranac grape skin extracts. While SCE offered a solvent-free approach with good •OH and O₂•⁻ scavenging, ethanol-assisted techniques (PLE and UAE) ignificantly improved polyphenol extraction and overall antioxidant capacity. These results highlight PLE, in particular, as a promising green strategy for recovering bioactive compounds from grape skin waste for nutraceutical, pharmaceutical, and cosmetic use.Tosti T, Dapčević A, Tešić Ž, editors. Book of abstrcats: 29th International Symposium on Separation Sciences: ISSS 2025; 2025 Sep 25-27; Belgrade, Serbia. Belgrade: Serbian Chemical Society; 2025. p. 51
Sperm motility parameters in common carp (Cyprinus carpio) using Computer-Assisted Sperm Analysis (CASA)
No full textThe common carp (Cyprinus carpio), a cyprinid species native to Eurasia, has been introduced worldwide
for food production, aquaculture, and recreational fishing, often becoming a dominant species in various
freshwater ecosystems. It is the third most widely cultivated and commercially significant freshwater fish
species globally (FAO 2013), with approximately 99.9% of the surface area of all fish farms in Serbia
dedicated to carp farming. Sperm motility directly affects reproductive success and the sustainability of fish
populations. Aquaculture, with its long and rich history, plays a crucial role in human nutrition and food
security, contributing significantly to economic development. In studies of male fertility, the most common
indicators of sperm quality are the computer-assisted sperm analysis (CASA) parameters of sperm
movement. In this study, ejaculate was collected from 10 individuals, in May, by abdominal massage from
tanks at the Centre for Fisheries and Applied Hydrobiology “Mali Dunav”, Faculty of Agriculture, University
of Belgrade, in sterile 15 ml plastic bottles. The body length of the analysed specimens ranged from 48 to
64 cm, while their weight ranged from 2.1 to 4.5 kg. Sperm was stored at 4°C and transported to the
laboratory, where activation was performed with water from the tanks. Spermatozoa motility was analyzed
using CASA. The total number of sperm analyzed ranged from 253 to 6,363. Total sperm motility (%)
among the analyzed specimens ranged from 55.79 to 84, while progressive motility (%) ranged from 53.30
to 83.65. These results indicate variability in motility parameters across individual specimens, with
consistent values for total and progressive motility. Sperm motility parameters are the most important
indicators for predicting the biological quality of male gametes and their effectiveness in fertilization trials.
Tracking sperm motility parameters in fish is essential for understanding reproductive health, improving
breeding success, and supporting the conservation and sustainable management of fish populations.Bosak S, Gračan R, Korać P, editors. Book of Abstracts: 15th Croatian Biological Congress: with international participation; 2025 Nov 20-23; Zagreb, Croatia. Zagreb: Croatian Biological Society; 2025. p. 257
Assessing the Chronic Effects of Dietary Aluminum on Fitness Traits, Acetylcholinesterase Activity, and Locomotion in Lymantria dispar L. Larvae
No full textAluminum is one of the most abundant elements in the Earth's crust. As Lepidoptera have not yet been used in studies describing the toxic impact of aluminium, the aim of this investigation was to identify the first visible changes in the development of Lymantria dispar larvae following dietary aluminum exposure and to assess whether this species is suitable as a potential model system for aluminum toxicity. In this study, the chronic effects of four metal concentrations on larvae from hatching to the fifth larval instar were investigated. We monitored changes in fitness traits (mass, relative growth rate, and days of development), acetylcholinesterase (AChE) activity, and locomotion parameters. L. dispar larvae were used as a model. Sublethal chronic dietary concentrations were 50, 250, 500, and 1000 µg Al/g dry food (T1, T2, T3, and T4, respectively). Our results show that chronic dietary aluminum exposure affects fitness traits and behavior, AChE activity, and locomotion
Mechanisms Maintaining Mitochondrial DNA Polymorphisms: The Role of Mito-Nuclear Interactions, Sex-Specific Selection, and Genotype-by-Environment Interactions in Drosophila subobscura
No full textSimple Summary
Drosophila subobscura is an interesting model to study forces that shape and maintain sympatric mitochondrial DNA (mtDNA) variation, due to the widespread presence of the two main, almost equally frequent haplotypes. Experimental setups using different life-history components enable us to study the adaptive significance of mtDNA variation and its effects on fitness while also trying to disentangle the role of different balancing selection mechanisms that operate in order to promote stable variation in natural populations. Constructing mito-nuclear experimental lines using backcrossing enables us to discern whether mito-nuclear interactions play a role in maintaining the aforementioned mtDNA variation. Since studies examining the maintenance of intrapopulation mitochondrial variability are scarce, our experimental results significantly contribute to this field of research.
Abstract
Experimental mito-nuclear introgression lines (MNILs) were established by backcrossing isofemale lines of D. subobscura originating from the same populations. MNILs were subjected to a series of life-history experiments designed to test the fitness of the bearers of different combinations of two main mtDNA haplotypes on their own nuclear background, as well as on the background of the opposite haplotype. By having 11 replicas of the four mito-nuclear combinations, we could test not only the adaptive significance of the differences between the two main haplotypes but also the influence of additional variation present within each of the 11 combinations on fitness. Testing the fitness of individuals of both sexes enabled us to examine if sex-specific selection has a role in maintaining the frequencies of the two mtDNA haplotypes in nature. Conducting the fitness assays on two different temperatures enabled us to test whether different temperatures favor specific mtDNA haplotypes or mito-nuclear genotypes and consequently promote stable sympatric mtDNA variation. The results show weak signature of genotype-by-environment interactions, and no sex-specific selection regarding differences between the two main haplotypes. However, individual models across different life-history components showed these two mechanisms at play in promoting mtDNA variability present in specific mito-nuclear crosses. Our models show that mito-nuclear interactions are, in fact, more important as units of selection