Delta opioid receptor on equine sperm cells: subcellular localization and involvement in sperm motility analyzed by computer assisted sperm analyzer (CASA)

<p>Abstract</p> <p>Background</p> <p>Opioid receptors and endogenous opioid peptides act not only in the control of nociceptive pathways, indeed several reports demonstrate the effects of opiates on sperm cell motility and morphology suggesting the importance of these receptors in the modulation of reproduction in mammals. In this study we investigated the expression of delta opioid receptors on equine spermatozoa by western blot/indirect immunofluorescence and its relationship with sperm cell physiology.</p> <p>Methods</p> <p>We analyzed viability, motility, capacitation, acrosome reaction and mitochondrial activity in the presence of naltrindole and DPDPE by means of a computer assisted sperm analyzer and a fluorescent confocal microscope. The evaluation of viability, capacitation and acrosome reaction was carried out by the double CTC/Hoechst staining, whereas mitochondrial activity was assessed by means of MitoTracker Orange dye.</p> <p>Results</p> <p>We showed that in equine sperm cells, delta opioid receptor is expressed as a doublet of 65 and 50 kDa molecular mass and is localized in the mid piece of tail; we also demonstrated that naltrindole, a delta opioid receptor antagonist, could be utilized in modulating several physiological parameters of the equine spermatozoon in a dose-dependent way. We also found that low concentrations of the antagonist increase sperm motility whereas high concentrations show the opposite effect. Moreover low concentrations hamper capacitation, acrosome reaction and viability even if the percentage of cells with active mitochondria seems to be increased; the opposite effect is exerted at high concentrations. We have also observed that the delta opioid receptor agonist DPDPE is scarcely involved in affecting the same parameters at the employed concentrations.</p> <p>Conclusions</p> <p>The results described in this paper add new important details in the comprehension of the mammalian sperm physiology and suggest new insights for improving reproduction and for optimizing equine breeding.</p

Functional Expression of the Extracellular Calcium Sensing Receptor (CaSR) in Equine Umbilical Cord Matrix Size-Sieved Stem Cells

The present study investigates the effects of high external calcium concentration ([Ca(2+)](o)) and the calcimimetic NPS R-467, a known calcium-sensing receptor (CaSR) agonist, on growth/proliferation of two equine size-sieved umbilical cord matrix mesenchymal stem cell (eUCM-MSC) lines. The involvement of CaSR on observed cell response was analyzed at both the mRNA and protein level.A large (>8 µm in diameter) and a small (<8 µm) cell line were cultured in medium containing: 1) low [Ca(2+)](o) (0.37 mM); 2) high [Ca(2+)](o) (2.87 mM); 3) NPS R-467 (3 µM) in presence of high [Ca(2+)](o) and 4) the CaSR antagonist NPS 2390 (10 µM for 30 min.) followed by incubation in presence of NPS R-467 in medium with high [Ca(2+)](o). Growth/proliferation rates were compared between groups. In large cells, the addition of NPS R-467 significantly increased cell growth whereas increasing [Ca(2+)](o) was not effective in this cell line. In small cells, both higher [Ca(2+)](o) and NPS R-467 increased cell growth. In both cell lines, preincubation with the CaSR antagonist NPS 2390 significantly inhibited the agonistic effect of NPS R-467. In both cell lines, increased [Ca(2+)](o) and/or NPS R-467 reduced doubling time values.Treatment with NPS R-467 down-regulated CaSR mRNA expression in both cell lines. In large cells, NPS R-467 reduced CaSR labeling in the cytosol and increased it at cortical level.In conclusion, calcium and the calcimimetic NPS R-467 reduce CaSR mRNA expression and stimulate cell growth/proliferation in eUCM-MSC. Their use as components of media for eUCM-MSC culture could be beneficial to obtain enough cells for down-stream purposes

Stress and mu opioid receptor in the management of gilthead sea bream (Sparus aurata) aquaculture

The growing consumption of aquaculture products requires always new techniques to increase the production yield. Generally, the intensification of aquaculture practices is associated with a stress level rise of bred fishes. Sensitivity to stress, leading to disease, reduced growth and mortality, is higher in larvae than in adult fish. The stress induced effects can be reduced acting on opiod receptors. In this light we evaluated the efficacy of naloxone, an opioid receptor antagonist, directly added to the water during Sparus aurata larval development. We found that in larvae subjected to artificial induced stressors, such as overcrowding, reduced pH, increased temperature and salinity, naloxone was useful to decrease the negative effects caused. In this Research highlight we discuss the finding of our recent study and research advancements

Impact of the severity of labour and birthplace on hormonal changes in the sheep

The onset and evolution of parturition are affected by environmental stressors, number of lambs, and dystocia. The regulation of parturition follows a chronological sequence, whose complete understanding is still rather complex. Progesterone (P4) is essential for ovine pregnancy maintenance. The placenta is the major source of P4, but also the fetus plays a critical role in the endocrine mechanisms that regulate the parturition timing. In the last 15 days before term, fetal adrenal gland increases cortisol (F) production which has two remarkable functions: firstly, it stimulates fetal lungs functional maturation and secondly in the placenta it enhances the expression of the steroidogenic enzyme P450c17, thus allowing P4 conversion to estrogens [1]. The correlation between maternal plasma F levels and the duration of labor as well as the correlation between pain intensity and plasma Betaendorphin (B-end) concentration have been studied [2]. Still, there are no reports of any correlation between these hormones, the severity of parturition pain and the environment. The aim of this study was therefore to determine whether, in sheep plasma concentrations of F, adrenaline (A), noradrenaline (NA), B-end, estradiol (E2) and P4 could be correlated to labor phase and intensity, and to the birthplace (sheepfold versus pasture). Sheep were well suited for handling. Animal care and use, as well as the experimental design of the study, were approved by the local animal ethics committee of the Department (DETO) (protocol n°1091/III/13 del 18/09/2017). The reproductive activity and pregnancy of 20 Comisana sheep (4-8 years old, multiparous) reared with a semi-wild system were monitored. Parturitions were classified in: eutocic, eutocic twin birth, dystocic and dystocic twin birth. Blood samples were taken from day 144 until labor and then during each stage of labor at the following time points: T0 (24 hrs before labor), T1 (prodromic stage), T2 (dilated stage), T3 (expulsive stage) T4 (stage of the fetal membrane expulsion), T5 (24 hrs after labor). Haematic concentrations of F, A, NA, B-end, E2 and P4 were evaluated by ELISA kits. Recorded data were then analyzed by SPSS software and statistical significance was set to P&lt;0.05. We found that 1) the kind of labor and the place in which it occurred affected significantly F, A, NA, B-end, E2 concentrations; 2) from T2 until T4 there was a significant increase of A and NA concentrations; 3) at T3 a significant increase of F concentration appeared; 4) B-end was the only compound whose concentration increased from T0 to T5; 5) dystocia induced significantly higher concentrations of all hormones related to stress. The present study shows that during labor a positive correlation between hormonal changes and phases/encountered difficulties exists (labor/F at T3 R=0.628; P=0.003 at T4 R=0.631; P=0.003 at T5 R=0.557; P=0.011: labor/A at T2 R=0.931; P=0.000; at T3 R=0.957; P=0.000: labor/NA at T1 R=0.470; P=0.037 at T2 R=0.982; P=0.000 at T3 R=0.956; P=0.000: labor/B-end at T0 R=0.788 P=0.000; at T1 R=0.562; P=0.10 at T2 R=0.885; P=0.000 at T3 R=0.929; P=0.000 at T4 R=0.720; P=0.000). Moreover, hormonal peaks were reached around the time of expulsion, suggesting that they could have been associated with muscle work, stress and pain. [1] Wood et al. Fetal and neonatal HPA Axis, Compr Physiol, 6(1): 33-62, 2015. [2] McMillen et al. Chronic stress – the key to parturition? Reprod Fertil Dev. 7(3): 499-507, 1995. 29

Expression of CYP27A1, CYP2R1 and VDR in equine cryptorchid testis

A functional vitamin D (VitD) requires a double step bioactivation by six cytochrome P450 (CYP) isoforms. The first step happens in the liver by four D-25-hydroxylase enzymes, among them the most active are CY27A1 and CYP2R1; the activation pathway ends in the kidney by a 1-α-hydroxylase [1]. The biological activity of VitD requires binding to the cytosolic VitD receptor (VDR), which translocate to the nucleus and act as a factor regulating the transcription of more than 200 genes modulating normal and cancer cell growth, differentiation, apoptosis, angiogenesis and metastatic potential [2]. VitD deficiency has been suggested as a risk factor for cancer because it impairs the anti-proliferative properties of vitamin D receptor (VDR) [3], and it adds to cryptorchidism as a cause of testicular cancer [4]. In the horse cryptorchidism is one of the male developmental defect that affects more than 9% of the subjects. Male reproductive tract expresses VDR and the enzymes involved in vitamin D activation through 25-hydroxilation [5]. Therefore, this study examined whether equine testis expresses CYP27A1 and/or CYP2R1 and VDR proteins and whether cryptorchidism may impair their expression thus enhancing the risk of developing testicular cancer. By western blot and immunohistochemistry, CYP27A1, CYP2R1 and VDR proteins were quantified and localized. Results demonstrated that all the three proteins were expressed in equine testis, moreover the expression level of CYP27A1 and VDR were significantly lower (P&lt;0.01 and P&lt;0.05 respectively) in the retained testis in respect to the contralateral scrotal testis. CYP2R1 protein resulted expressed at the same level both in the undescended and in the scrotal testis. This study showed that also in the horse testes play a role in the vitamin D metabolism

A case of suspected eosinophilic myocarditis recognized by a fully noninvasive approach and safely treated with corticosteroids despite an underling Hepatitis C virus-hepatitis

Hypereosinophilic syndrome can lead to acute myocarditis with a potentially severe systolic dysfunction and serious complications. A 75-year-old patient suffering from Hepatitis C virus (HCV) related-hepatitis came to our observation for idiopatic hypereosinophilic syndrome and acute severe cardiac systolic dysfunction without coronaropathy. Cardiac magnetic resonance showed a 'patchy' subendocardial and intramyocardial late gadolinium enhancement pattern often seen in eosinophilic myocarditis (EM). Assuming EM, appropriate corticosteroid therapy was initiated and it led to clinical remission. Despite endomyocardial biopsy&nbsp;(EMB) is the diagnostic gold standard for EM, in this case only a noninvasive integrated imaging approach was successfully attempted. Given an adequate clinical context, in our opinion EM can be correctly recognized without EMB and so promptly and safely treated with corticosteroids, even when an underling mild HCV-hepatitis is present

Male reproductive physiology of Paracentrotus lividus: monthly and seasonal evaluations to study the effects of the environment on gonadal development and sperm cells kinetic

Paracentrotus lividus is a model species for research in different areas, it is also important for modern aquaculture interested in breeding innovative species for greater product diversifications and reduction of the wild stock fishing pressure. This Echinoid was considered a seasonal niche product in the past; nowadays it is highly required on the market for the nutritional characteristics (high protein and low fat) of gonads, the edible part. Our work aims at optimizing farm technics by the study of the habitat impact on P. lividus male reproductive physiology. We correlated environmental variables of the fishing area (temperature, humidity, photoperiod, water temperature, moon phases) to reproductive parameters (morphological gonadal development, sperm cells kinetic). We analyzed data to highlight the relevance, for reproduction, of the length (month or season) of the observations. The significance of this work to the field of aquatic sciences is to drive aquaculture facilities in choosing the desirable period to recover the best spermatozoa with highest total and progressive motility. Moreover setting useful parameters to mimic the best environmental conditions, the highest gonadal development could be reached. This could also promote gametes exchanges between aquaculture facilities in the world taking advantage of the environment dependent gametes maturity