Isolation and characterization of mammary epithelial cells derived from Göttingen Minipigs: A comparative study versus hybrid pig cells from the IMI-ConcePTION Project.

The value of pig as "large animal model" is a well-known tool for translational medicine, but it can also be beneficial in studying animal health in a one-health vision. The ConcePTION Project aims to provide new information about the risks associated with medication use during breastfeeding, as this information is not available for most commonly used drugs. In the IMI-Conception context, Göttingen Minipigs have been preferred to hybrid pigs for their genetic stability and microbiological control. For the first time, in the present research, three primary cell cultures of mammary epithelial cells were isolated and characterized from Göttingen Minipigs (mpMECs), including their ability to create the epithelial barrier. In addition, a comparative analysis between Göttingen Minipigs and commercial hybrid pig mammary epithelial cells (pMECs) was conducted. Epithelial markers: CKs, CK18, E-CAD, ZO-1 and OCL, were expressed in both mpMECs and pMECs. RT2 Profiler PCR Array Pig Drug Transporters showed a similar profile in mRNA drug transporters. No difference in energy production under basal metabolic condition was evidenced, while under stressed state, a different metabolic behaviour was shown between mpMECs vs pMECs. TEER measurement and sodium fluorescein transport, indicated that mpMECs were able to create an epithelial barrier, although, this turned out to be less compact than pMECs. By comparing mpMECs with mammary epithelial cells isolated from Hybrid pigs (pMECs), although both cell lines have morphological and phenotypic characteristics that make them both useful in barrier studies, some specific differences exist and must be considered in a translational perspective.

Extracellular cAMP and MRP4 activity influence in vitro capacitation and fertilizing ability of pig spermatozoa.

cAMP has been reported to be an essential driver of sperm capacitation. In bovine sperm cAMP efflux through multidrug resistance-associated protein 4 (MRP4) has been suggested to maintain intracellular cAMP homeostasis and generate extracellular signaling able to regulate capacitation. The aim of this work was to determine whether extracellular cAMP may influence in vitro pig sperm capacitation and acquisition of fertilizing ability and to evaluate the role of MRP4. In vitro sperm capacitation and gamete coincubation were performed in Brackett and Oliphant's medium (BO) in presence of caffeine (Ctr+) or in BO without caffeine (Ctr-) supplemented with 0, 8, 9, 10 mM cAMP. Despite the percentage of capacitated sperm, assayed by immunolocalization of tyrosine-phosphorylated proteins, was significantly lower in Ctr- compared to Ctr+, it increased supplementing 10 mM cAMP to Ctr- reaching values similar to Ctr+. The absence of caffeine during gamete coincubation reduced the fertilization rate compared to Ctr+, while 10 mM cAMP supplementation to Ctr- increased the fertilization rate reaching values similar to Ctr + . The presence of MRP4 in pig spermatozoa was detected for the first time by western blot and immunohistochemistry assays. To evaluate MRP4 role on pig sperm capacitation, in vitro capacitation and gamete coincubation were performed in Ctr + in presence of MK571, a MRP4 selective inhibitor. MK571 reduced the percentage of capacitated cells and the fertilization rate, while cAMP addition fully reversed MRP4 blockade consequences. Present findings suggest that, under our in vitro conditions, extracellular cAMP and MRP4 activity influence pig sperm capacitating events.

Phloem and Xylem Responses Are Both Implicated in Huanglongbing Tolerance of Sugar Belle.

Although huanglongbing (HLB) is a devastating citrus disease, improved tolerant cultivars, such as Sugar Belle (SB) mandarin, have been identified. To understand the responses that HLB-affected SB undergoes, we compared 14CO2 fixation, carbohydrate export, phloem callose accumulation, relative expression of plant defense activators, and anatomical changes between healthy and infected SB trees versus susceptible Pineapple (PA) sweet orange. Eight- to ten-week-old leaves of infected SB showed a 2.5-fold increase in 14CO2 fixation and a 13% decrease in 14C-carbohydrate export, whereas HLB-affected PA presented a decrease of 33 and 50%, respectively. The mean distance of a callose deposit to its closest neighbor was 36% smaller in infected SB versus healthy, whereas in HLB-affected PA, it was 33% higher. Expression of papain-like cysteine proteases (PLCPs) was upregulated in SB but downregulated in PA. Infected SB showed minor alterations in the number of xylem vessels, a 16% larger xylem vessel lumen area, and a 14% increase in the proportional area of the xylem. In contrast, PA showed a 2.4-fold increase in the xylem vessel number and a 2% increase in the proportional xylem area. Three complementary mechanisms of tolerance in SB are hypothesized: (i) increased carbohydrate availability induced by greater CO2 fixation, mild effect in carbohydrate export, and local accumulation of callose in the phloem; (ii) activation of defense response via upregulation of PLCPs, and (iii) increased investment in the xylem structure. Thus, phloem and xylem modifications seem to be involved in SB tolerance.

Evaluation of the Epithelial Barrier Integrity in Primary Cultures of Pig Mammary Epithelial Cells.

A major feature of epithelial and endothelial cells is the creation of biological barriers able to protect the body against stressors that could compromise homeostasis. The ability to characterize biological barriers in vitro is an important study tool especially used for the intestinal barrier, the blood-brain barrier, and the lung barrier. The strength and integrity of biological barriers may be assessed by the measurement of the transepithelial/transendothelial electrical resistance (TEER) that reflects the ionic conductance of the paracellular pathway. The TEER measurement is a quantitative, non-invasive, highly useful, and representative method that must be strictly standardized. Here we describe a quantitative protocol to assess the mammary epithelial barrier integrity by combining the TEER measurement with a test for studying the passage of the sodium fluorescein, that is, a hydrophilic paracellular marker. Being the swine species an excellent translational model, primary cultures of mammary epithelial cells, isolated from hybrid pig tissue collected at slaughterhouse, are used.

Mineralocorticoid Receptor Antagonism Prevents Type 2 Familial Partial Lipodystrophy Brown Adipocyte Dysfunction.

Type-2 Familial Partial Lipodystrophy (FPLD2), a rare lipodystrophy caused by LMNA mutations, is characterized by a loss of subcutaneous fat from the trunk and limbs and excess accumulation of adipose tissue in the neck and face. Several studies have reported that the mineralocorticoid receptor (MR) plays an essential role in adipose tissue differentiation and functionality. We previously showed that brown preadipocytes isolated from a FPLD2 patient's neck aberrantly differentiate towards the white lineage. As this condition may be related to MR activation, we suspected altered MR dynamics in FPLD2. Despite cytoplasmic MR localization in control brown adipocytes, retention of MR was observed in FPLD2 brown adipocyte nuclei. Moreover, overexpression of wild-type or mutated prelamin A caused GFP-MR recruitment to the nuclear envelope in HEK293 cells, while drug-induced prelamin A co-localized with endogenous MR in human preadipocytes. Based on in silico analysis and in situ protein ligation assays, we could suggest an interaction between prelamin A and MR, which appears to be inhibited by mineralocorticoid receptor antagonism. Importantly, the MR antagonist spironolactone redirected FPLD2 preadipocyte differentiation towards the brown lineage, avoiding the formation of enlarged and dysmorphic lipid droplets. Finally, beneficial effects on brown adipose tissue activity were observed in an FPLD2 patient undergoing spironolactone treatment. These findings identify MR as a new lamin A interactor and a new player in lamin A-linked lipodystrophies.

Isolation of Vascular Wall Mesenchymal Stem Cells from the Thoracic Aorta of Adult Göttingen Minipigs: A New Protocol for the Simultaneous Endothelial Cell Collection.

Two main classes of perivascular multipotent populations have been described: the microvascular pericytes and the vascular wall mesenchymal stem cells (VW-MSCs). VW-MSCs are isolated from large vessels in many species and they participate in vascular remodeling together with other cellular components such as endothelial cells. Considering that the Göttingen Minipigs are widely used in Europe as a translational model in the field of cardiovascular diseases, the aim of the present research was to isolate VW-MSCs from the adult aorta of Göttingen Minipigs while preserving and also collecting endothelial cells. The results obtained in the present research demonstrated that this new protocol allows us to obtain a pure population of VW-MSCs and endothelial cells. VW-MSCs from Göttingen Minipigs responded fully to the MSC minima international criteria, being positive to CD105, CD90, and CD44 and negative to CD45 and CD34. Moreover, VW-MSCs presented a differentiative potential towards osteogenic, chondrogenic, and adipogenic lineages. Overall, the present protocol, preserving the viability and phenotypic features of the two isolated populations, opens future possibilities of using minipig VW-MSCs and endothelial cells in in vitro vascular remodeling studies.

The sieve-element endoplasmic reticulum: A focal point of phytoplasma-host plant interaction?

The rough endoplasmic reticulum (r-ER) is of paramount importance for adaptive responses to biotic stresses due to an increased demand for de novo synthesis of immunity-related proteins and signaling components. In nucleate cells, disturbance of r-ER integrity and functionality leads to the "unfolded protein response" (UPR), which is an important component of innate plant immune signalling. In contrast to an abundance of reports on r-ER responses to biotic challenges, sieve-element endoplasmic reticulum (SE-ER) responses to phytoplasma infection have not been investigated. We found that morphological SE-ER changes, associated with phytoplasma infection, are accompanied by differential expression of genes encoding proteins involved in shaping and anchoring the reticulum. Phytoplasma infection also triggers an increased release of bZIP signals from the (SE-ER)/r-ER and consequent differential expression of UPR-related genes. The modified expression patterns seem to reflect a trade-off between survival of host cells, needed for the phytoplasmic biotrophic lifestyle, and phytoplasmas. Specialized plasmodesmata between sieve element and companion cell may provide a corridor for transfer of phytoplasma effectors inducing UPR-related gene expression in companion cells.

Can masked gaze and arrow stimuli elicit overt orienting of attention? A registered report.

Viewing an averted gaze can elicit saccades towards the corresponding location. Here, the automaticity of this gaze-following behaviour phenomenon was further tested by exploring whether such an effect can be detected in response to briefly-presented masked averted gazes. Participants completed an oculomotor interference task consisting of making leftward/rightward saccades according to a symbolic instruction cue. Crucially, either a task-irrelevant averted-gaze face or an arrow (i.e., a non-social control stimulus) was also presented in different blocks of trials. Faces and arrows were presented for either 1000 ms, or 8 ms and then backward-masked, to reduce the likelihood of conscious processing. Worse oculomotor performance emerged when the saccade direction did not match (vs match) that suggested by the task-irrelevant gaze/arrow stimuli in the unmasked condition. However, in the masked condition, no oculomotor interference occurred for any task-irrelevant stimulus. Results enrich knowledge about boundary conditions for gaze/arrow-driven orienting using ecological attention measures.

1,5-disubstituted-1,2,3-triazoles counteract mitochondrial dysfunction acting on F1FO-ATPase in models of cardiovascular diseases.

The compromised viability and function of cardiovascular cells are rescued by small molecules of triazole derivatives (Tzs), identified as 3a and 3b, by preventing mitochondrial dysfunction. The oxidative phosphorylation improves the respiratory control rate in the presence of Tzs independently of the substrates that energize the mitochondria. The F1FO-ATPase, the main candidate in mitochondrial permeability transition pore (mPTP) formation, is the biological target of Tzs and hydrophilic F1 domain of the enzyme is depicted as the binding region of Tzs. The protective effect of Tz molecules on isolated mitochondria was corroborated by immortalized cardiomyocytes results. Indeed, mPTP opening was attenuated in response to ionomycin. Consequently, increased mitochondrial roundness and reduction of both length and interconnections between mitochondria. In in-vitro and ex-vivo models of cardiovascular pathologies (i.e., hypoxia-reoxygenation and hypertension) were used to evaluate the Tzs cardioprotective action. Key parameters of porcine aortic endothelial cells (pAECs) oxidative metabolism and cell viability were not affected by Tzs. However, in the presence of either 1 µM 3a or 0.5 µM 3b the impaired cell metabolism of pAECs injured by hypoxia-reoxygenation was restored to control respiratory profile. Moreover, endothelial cells isolated from SHRSP exposed to high-salt treatment rescued the Complex I activity and the endothelial capability to form vessel-like tubes and vascular function in presence of Tzs. As a result, the specific biochemical mechanism of Tzs to block Ca2+-activated F1FO-ATPase protected cell viability and preserved the pAECs bioenergetic metabolism upon hypoxia-reoxygenation injury. Moreover, SHRSP improved vascular dysfunction in response to a high-salt treatment.

Poor shoot and leaf growth in Huanglongbing-affected sweet orange is associated with increased investment in defenses.

Citrus disease Huanglongbing (HLB) causes sparse (thinner) canopies due to reduced leaf and shoot biomass. Herein, we present results demonstrating the possible mechanisms behind compromised leaf growth of HLB-affected 'Valencia' sweet orange trees by comparing morphological, transcriptome, and phytohormone profiles at different leaf development phases (1. buds at the start of the experiment; 2. buds on day 5; . 3. leaf emergence; 4. leaf expansion; and 5. leaf maturation) to healthy trees. Over a period of 3 months (in greenhouse conditions), HLB-affected trees had ≈40% reduction in growth traits such as tree height, number of shoots per tree, shoot length, internode length, and leaf size compared to healthy trees. In addition, buds from HLB-affected trees lagged by ≈1 week in sprouting as well as leaf growth. Throughout the leaf development, high accumulation of defense hormones, salicylic acid (SA) and abscisic acid (ABA), and low levels of growth-promoting hormone (auxin) were found in HLB-affected trees compared to healthy trees. Concomitantly, HLB-affected trees had upregulated differentially expressed genes (DEGs) encoding SA, ABA, and ethylene-related proteins in comparison to healthy trees. The total number of cells per leaf was lower in HLB-affected trees compared to healthy trees, which suggests that reduced cell division may coincide with low levels of growth-promoting hormones leading to small leaf size. Both bud dieback and leaf drop were higher in HLB-affected trees than in healthy trees, with concomitant upregulated DEGs encoding senescence-related proteins in HLB-affected trees that possibly resulted in accelerated aging and cell death. Taken together, it can be concluded that HLB-affected trees had a higher tradeoff of resources on defense over growth, leading to sparse canopies and a high tree mortality rate with HLB progression.

Anticancer activity of an Artemisia annua L. hydroalcoholic extract on canine osteosarcoma cell lines.

Since ancient times, Artemisia annua (A. annua) has been used as a medicinal plant in Traditional Chinese Medicine. In addition, recent studies have investigated the cytotoxic effects of A. annua extracts towards cancer cells. The leading aim of the present research is to evaluate the cytotoxic effects of an hydroalcoholic extract of A. annua on two canine osteosarcoma (OSA) cell lines, OSCA-8 and OSCA-40, focusing on the possible involvement of ferroptosis. The quantitative determination of artemisinin concentration in the extract, culture medium and OSA cells was carried out through the use of an instrumental analytical method based on liquid chromatography coupled with spectrophotometric detection and tandem mass spectrometry (LC-DAD-MS/MS). OSCA-8 and OSCA-40 were exposed to different dilutions of the extract for the EC50 calculation then the uptake of artemisinin by the cells, the effects on the cell cycle, the intracellular iron level, the cellular morphology and the lipid oxidation state were evaluated. A concentration of artemisinin of 63.8 ± 3.4 µg/mL was detected in the extract. A dose-dependent cytotoxic effect was evidenced. In OSCA-40 alterations of the cell cycle and a significantly higher intracellular iron content were observed. In both cell lines the treatment with the extract was associated with lipid peroxidation and with the appearance of a "ballooning" phenotype suggesting the activation of ferroptosis. In conclusion the A. annua idroalcoholic extract utilized in this study showed anticancer activity on canine OSA cell lines that could be useful in treating drug resistant canine OSAs.

Cellular Metabolism and Bioenergetic Function in Human Fibroblasts and Preadipocytes of Type 2 Familial Partial Lipodystrophy.

LMNA mutation is associated with type-2 familial partial lipodystrophy (FPLD2). The disease causes a disorder characterized by anomalous accumulation of body fat in humans. The dysfunction at the molecular level is triggered by a lamin A/C mutation, impairing the cell metabolism. In human fibroblasts and preadipocytes, a trend for ATP production, mainly supported by mitochondrial oxidative metabolism, is detected. Moreover, primary cell lines with FPLD2 mutation decrease the mitochondrial ATP production if compared with the control, even if no differences are observed in the oxygen consumption rate of bioenergetic parameters (i.e., basal and maximal respiration, spare respiratory capacity, and ATP turnover). Conversely, glycolysis is only inhibited in FPLD2 fibroblast cell lines. We notice that the amount of ATP produced in the fibroblasts is higher than in the preadipocytes, and likewise in the control, with respect to FPLD2, due to a more active oxidative phosphorylation (OXPHOS) and glycolysis. Moreover, the proton leak parameter, which characterizes the transformation of white adipose tissue to brown/beige adipose tissue, is unaffected by FPLD2 mutation. The metabolic profile of fibroblasts and preadipocytes is confirmed by the ability of these cell lines to increase the metabolic potential of both OXPHOS and glycolysis under energy required independently by the FPLD2 mutation.

The Impairment of Cell Metabolism by Cardiovascular Toxicity of Doxorubicin Is Reversed by Bergamot Polyphenolic Fraction Treatment in Endothelial Cells.

The beneficial effects of bergamot polyphenolic fraction (BPF) on the mitochondrial bioenergetics of porcine aortic endothelial cells (pAECs) were verified under the cardiotoxic action of doxorubicin (DOX). The cell viability of pAECs treated for 24 h with different concentrations of DOX was reduced by 50%, but the negative effect of DOX was reversed in the presence of increasing doses of BPF (100 µg/mL and 200 µg/mL BPF). An analysis of the protective effect of BPF on the toxic action of DOX was also carried out on cell respiration. We observed the inhibition of the mitochondrial activity at 10 µM DOX, which was not restored by 200 µg/mL BPF. Conversely, the decrease in basal respiration and ATP production caused by 0.5 or 1.0 µM DOX were improved in the presence of 100 or 200 µg/mL BPF, respectively. After 24 h of cell recovery with 100 µg/mL or 200 µg/mL BPF on pAECs treated with 0.5 µM or 1.0 µM DOX, respectively, the mitochondrial parameters of oxidative metabolism impaired by DOX were re-boosted.

Increased susceptibility to Chrysanthemum Yellows phytoplasma infection in Atcals7ko plants is accompanied by enhanced expression of carbohydrate transporters.

MAIN CONCLUSION: Loss of CALS7 appears to confer increased susceptibility to phytoplasma infection in Arabidopsis, altering expression of genes involved in sugar metabolism and membrane transport. Callose deposition around sieve pores, under control of callose synthase 7 (CALS7), has been interpreted as a mechanical response to limit pathogen spread in phytoplasma-infected plants. Wild-type and Atcals7ko mutants were, therefore, employed to unveil the mode of involvement of CALS7 in the plant's response to phytoplasma infection. The fresh weights of healthy and CY-(Chrysanthemum Yellows) phytoplasma-infected Arabidopsis wild type and mutant plants indicated two superimposed effects of the absence of CALS7: a partial impairment of photo-assimilate transport and a stimulated phytoplasma proliferation as illustrated by a significantly increased phytoplasma titre in Atcal7ko mutants. Further studies solely dealt with the effects of CALS7 absence on phytoplasma growth. Phytoplasma infection affected sieve-element substructure to a larger extent in mutants than in wild-type plants, which was also true for the levels of some free carbohydrates. Moreover, infection induced a similar upregulation of gene expression of enzymes involved in sucrose cleavage (AtSUS5, AtSUS6) and transmembrane transport (AtSWEET11) in mutants and wild-type plants, but an increased gene expression of carbohydrate transmembrane transporters (AtSWEET12, AtSTP13, AtSUC3) in infected mutants only. It remains still unclear how the absence of AtCALS7 leads to gene upregulation and how an increased intercellular mobility of carbohydrates and possibly effectors contributes to a higher susceptibility. It is also unclear if modified sieve-pore structures in mutants allow a better spread of phytoplasmas giving rise to higher titre.

Mitochondria Bioenergetic Functions and Cell Metabolism Are Modulated by the Bergamot Polyphenolic Fraction.

The bergamot polyphenolic fraction (BPF) was evaluated in the F1FO-ATPase activity of swine heart mitochondria. In the presence of a concentration higher than 50 µg/mL BPF, the ATPase activity of F1FO-ATPase, dependent on the natural cofactor Mg2+, increased by 15%, whereas the enzyme activity in the presence of Ca2+ was inhibited by 10%. By considering this opposite BPF effect, the F1FO-ATPase activity involved in providing ATP synthesis in oxidative phosphorylation and triggering mitochondrial permeability transition pore (mPTP) formation has been evaluated. The BPF improved the catalytic coupling of oxidative phosphorylation in the presence of a substrate at the first phosphorylation site, boosting the respiratory control ratios (state 3/state 4) by 25% and 85% with 50 µg/mL and 100 µg/mL BPF, respectively. Conversely, the substrate at the second phosphorylation site led to the improvement of the state 3/state 4 ratios by 15% only with 100 µg/mL BPF. Moreover, the BPF carried out its beneficial effect on the mPTP phenomenon by desensitizing the pore opening. The acute effect of the BPF on the metabolism of porcine aortica endothelial cells (pAECs) showed an ATP rate index greater than one, which points out a prevailing mitochondrial oxidative metabolism with respect to the glycolytic pathway, and this ratio rose by about three times with 100 µg/mL BPF. Consistently, the mitochondrial ATP turnover, in addition to the basal and maximal respiration, were higher in the presence of the BPF than in the controls, and the MTT test revealed an increase in cell viability with a BPF concentration above 200 µg/mL. Therefore, the molecule mixture of the BPF aims to ensure good performance of the mitochondrial bioenergetic parameters.

Efficacy of Stem Cell Therapy in Large Animal Models of Ischemic Cardiomyopathies: A Systematic Review and Meta-Analysis.

Stem-cell therapy provides a promising strategy for patients with ischemic heart disease. In recent years, numerous studies related to this therapeutic approach were performed; however, the results were often heterogeneous and contradictory. For this reason, we conducted a systematic review and meta-analysis of trials, reporting the use of stem-cell treatment against acute or chronic ischemic cardiomyopathies in large animal models with regard to Left Ventricular Ejection Fraction (LVEF). The defined research strategy was applied to the PubMed database to identify relevant studies published from January 2011 to July 2021. A random-effect meta-analysis was performed on LVEF mean data at follow-up between control and stem-cell-treated animals. In order to improve the definition of the effect measure and to analyze the factors that could influence the outcomes, a subgroup comparison was conducted. Sixty-six studies (n = 1183 animals) satisfied our inclusion criteria. Ischemia/reperfusion infarction was performed in 37 studies, and chronic occlusion in 29 studies; moreover, 58 studies were on a pig animal model. The meta-analysis showed that cell therapy increased LVEF by 7.41% (95% Confidence Interval 6.23−8.59%; p < 0.001) at follow-up, with significative heterogeneity and high inconsistency (I2 = 82%, p < 0.001). By subgroup comparison, the follow-up after 31−60 days (p = 0.025), the late cell injection (>7 days, p = 0.005) and the route of cellular delivery by surgical treatment (p < 0.001) were significant predictors of LVEF improvement. This meta-analysis showed that stem-cell therapy may improve heart function in large animal models and that the swine specie is confirmed as a relevant animal model in the cardiovascular field. Due to the significative heterogeneity and high inconsistency, future translational studies should be designed to take into account the evidenced predictors to allow for the reduction of the number of animals used.

Development of a Pig Mammary Epithelial Cell Culture Model as a Non-Clinical Tool for Studying Epithelial Barrier-A Contribution from the IMI-ConcePTION Project.

The ConcePTION project aims at generating further knowledge about the risks related to the use of medication during breastfeeding, as this information is lacking for most commonly used drugs. Taking into consideration multiple aspects, the pig model has been considered by the consortium as the most appropriate choice. The present research was planned to develop an efficient method for the isolation and culture of porcine Mammary Epithelial Cells (pMECs) to study the mammary epithelial barrier in vitro. Mammary gland tissues were collected at a local slaughterhouse, dissociated and the selected cellular population was cultured, expanded and characterized by morphology, cell cycle analysis and immunophenotyping. Their ability to create a barrier was tested by TEER measurement and sodium fluorescein transport activity. Expression of 84 genes related to drug transporters was evaluated by a PCR array. Our results show that primary cells express epithelial cell markers: CKs, CK18, E-Cad and tight junctions molecules ZO-1 and OCL. All the three pMEC cellular lines were able to create a tight barrier, although with different strengths and kinetics, and express the main ABC and SLC drug transporters. In conclusion, in the present paper we have reported an efficient method to obtain primary pMEC lines to study epithelial barrier function in the pig model.

Testicular Melatonin and Its Pathway in Roe Deer Bucks (Capreolus capreolus) during Pre- and Post-Rut Periods: Correlation with Testicular Involution.

Roe deer are seasonal breeders with a complete yearly testicular cycle. The peak in reproductive activity is recorded during summer, the rutting period, with the highest levels of androgens and testicular weight. Melatonin plays a pivotal role in seasonal breeders by stimulating the hypothalamus-pituitary-gonads axis and acting locally; in different species, its synthesis within testes has been reported. The aim of this study was to evaluate the physiological melatonin pattern within roe deer testes by comparing data obtained from animals sampled during pre- and post-rut periods. Melatonin was quantified in testicular parenchyma, along with the genetic expression of enzymes involved in its local synthesis (AANAT and ASMT) and function (UCP1). Melatonin receptors, MT1-2, were quantified both at protein and gene expression levels. Finally, to assess changes in reproductive hormonal profiles, testicular dehydroepiandrosterone (DHEA) was quantified and used for a correlation analysis. Melatonin and AANAT were detected in all samples, without significant differences between pre- and post-rut periods. Despite DHEA levels confirming testicular involution during the post-rut period, no correlations appeared between such involution and melatonin pathways. This study represents the first report regarding melatonin synthesis in roe deer testes, opening the way for future prospective studies in the physiology of this species.

Vitamin K Vitamers Differently Affect Energy Metabolism in IPEC-J2 Cells.

The fat-soluble vitamin K (VK) has long been known as a requirement for blood coagulation, but like other vitamins, has been recently recognized to play further physiological roles, particularly in cell development and homeostasis. Vertebrates cannot de novo synthesize VK, which is essential, and it can only be obtained from the diet or by the activity of the gut microbiota. The IPEC-J2 cell line, obtained from porcine small intestine, which shows strong similarities to the human one, represents an excellent functional model to in vitro study the effect of compounds at the intestinal level. The acute VK treatments on the bioenergetic features of IPEC-J2 cells were evaluated by Seahorse XP Agilent technology. VK exists in different structurally related forms (vitamers), all featured by a naphtoquinone moiety, but with distinct effects on IPEC-J2 energy metabolism. The VK1, which has a long hydrocarbon chain, at both concentrations (5 and 10 µM), increases the cellular ATP production due to oxidative phosphorylation (OXPHOS) by 5% and by 30% through glycolysis. The VK2 at 5 µM only stimulates ATP production by OXPHOS. Conversely, 10 µM VK3, which lacks the long side chain, inhibits OXPHOS by 30% and glycolysis by 45%. However, even if IPEC-J2 cells mainly prefer OXPHOS to glycolysis to produce ATP, the OXPHOS/glycolysis ratio significantly decreases in VK1-treated cells, is unaffected by VK2, and only significantly increased by 10 µM VK3. VK1, at the two concentrations tested, does not affect the mitochondrial bioenergetic parameters, while 5 µM VK2 increases and 5 µM VK3 reduces the mitochondrial respiration (i.e., maximal respiration and spare respiratory capacity). Moreover, 10 µM VK3 impairs OXPHOS, as shown by the increase in the proton leak, namely the proton backward entry to the matrix space, thus pointing out mitochondrial toxicity. Furthermore, in the presence of both VK1 and VK2 concentrations, the glycolytic parameters, namely the glycolytic capacity and the glycolytic reserve, are unaltered. In contrast, the inhibition of glycoATP production by VK3 is linked to the 80% inhibition of glycolysis, resulting in a reduced glycolytic capacity and reserve. These data, which demonstrate the VK ability to differently modulate IPEC-J2 cell energy metabolism according to the different structural features of the vitamers, can mirror VK modulatory effects on the cell membrane features and, as a cascade, on the epithelial cell properties and gut functions: balance of salt and water, macromolecule cleavage, detoxification of harmful compounds, and nitrogen recycling.