Presence and Function of Dopamine Transporter (DAT) in Stallion Sperm : Dopamine Modulates Sperm Motility and Acrosomal Integrity

Dopamine is a catecholamine with multiple physiological functions, playing a key role in nervous system; however its participation in reproductive processes and sperm physiology is controversial. High dopamine concentrations have been reported in different portions of the feminine and masculine reproductive tract, although the role fulfilled by this catecholamine in reproductive physiology is as yet unknown. We have previously shown that dopamine type 2 receptor is functional in boar sperm, suggesting that dopamine acts as a physiological modulator of sperm viability, capacitation and motility. In the present study, using immunodetection methods, we revealed the presence of several proteins important for the dopamine uptake and signalling in mammalian sperm, specifically monoamine transporters as dopamine (DAT), serotonin (SERT) and norepinephrine (NET) transporters in equine sperm. We also demonstrated for the first time in equine sperm a functional dopamine transporter using 4-[4-(Dimethylamino)styryl]-N-methylpyridinium iodide (ASP+ ), as substrate. In addition, we also showed that dopamine (1 mM) treatment in vitro, does not affect sperm viability but decreases total and progressive sperm motility. This effect is reversed by blocking the dopamine transporter with the selective inhibitor vanoxerine (GBR12909) and non-selective inhibitors of dopamine reuptake such as nomifensine and bupropion. The effect of dopamine in sperm physiology was evaluated and we demonstrated that acrosome integrity and thyrosine phosphorylation in equine sperm is significantly reduced at high concentrations of this catecholamine. In summary, our results revealed the presence of monoamine transporter DAT, NET and SERT in equine sperm, and that the dopamine uptake by DAT can regulate sperm function, specifically acrosomal integrity and sperm motility

Spatial distribution analysis with capture effect of a mobile S-ALOHA network

The throughput performance of a mobile S-ALOHA network can be improved considering the capture phenomenon, which also depends on the spatial distribution of the mobiles within the cell. We have studied the capture probabilities that arise in a mobile radio scenario in the presence of fading and shadowing, and considering both, uniform and non-uniform spatial distribution models. In particular we were interested in the limit behaviour of these models, which has been shown to be directly related to the capture probability. This analysis allows a quantitative comparison of three spatial distribution models for mobile users under real mobile channels. The use of an exponential backoff retransmission algorithm is considered. With these assumptions the performance of the anarchic ALOHA is improved considerably obtaining higher throughput values with stabilised behaviour and lower delays values.Peer ReviewedPostprint (published version

Spatial distribution analysis with capture effect of a mobile S-ALOHA network

The throughput performance of a mobile S-ALOHA network can be improved considering the capture phenomenon, which also depends on the spatial distribution of the mobiles within the cell. We have studied the capture probabilities that arise in a mobile radio scenario in the presence of fading and shadowing, and considering both, uniform and non-uniform spatial distribution models. In particular we were interested in the limit behaviour of these models, which has been shown to be directly related to the capture probability. This analysis allows a quantitative comparison of three spatial distribution models for mobile users under real mobile channels. The use of an exponential backoff retransmission algorithm is considered. With these assumptions the performance of the anarchic ALOHA is improved considerably obtaining higher throughput values with stabilised behaviour and lower delays values.Peer Reviewe

Synergistic combination therapy delivered via layer‐by‐layer nanoparticles induces solid tumor regression of ovarian cancer

Abstract The majority of patients with high grade serous ovarian cancer (HGSOC) develop recurrent disease and chemotherapy resistance. To identify drug combinations that would be effective in treatment of chemotherapy resistant disease, we examined the efficacy of drug combinations that target the three antiapoptotic proteins most commonly expressed in HGSOC—BCL2, BCL‐XL, and MCL1. Co‐inhibition of BCL2 and BCL‐XL (ABT‐263) with inhibition of MCL1 (S63845) induces potent synergistic cytotoxicity in multiple HGSOC models. Since this drug combination is predicted to be toxic to patients due to the known clinical morbidities of each drug, we developed layer‐by‐layer nanoparticles (LbL NPs) that co‐encapsulate these inhibitors in order to target HGSOC tumor cells and reduce systemic toxicities. We show that the LbL NPs can be designed to have high association with specific ovarian tumor cell types targeted in these studies, thus enabling a more selective uptake when delivered via intraperitoneal injection. Treatment with these LbL NPs displayed better potency than free drugs in vitro and resulted in near‐complete elimination of solid tumor metastases of ovarian cancer xenografts. Thus, these results support the exploration of LbL NPs as a strategy to deliver potent drug combinations to recurrent HGSOC. While these findings are described for co‐encapsulation of a BCL2/XL and a MCL1 inhibitor, the modular nature of LbL assembly provides flexibility in the range of therapies that can be incorporated, making LbL NPs an adaptable vehicle for delivery of additional combinations of pathway inhibitors and other oncology drugs

Dopamine transporter inactivation attenuates the inhibitory effect of 1 mM dopamine on total sperm motility.

<p>Equine sperm were incubated at 37°C with 1 mM dopamine, with and without the specific DAT inhibitor, 10 µM GBR12909 for 1 hour, in Withenn’s buffer. Total motility was calculated using the CASA system, with a minimum of 500 sperm analyzed per experiment. Total motility was normalized to time zero of the control. Results are the mean ± SEM of four independent experiments. The different letters show significant changes between each treatment with p<0.01. We have considered in each experiment a control to normalize the results to the basal sperm condition before the incubation with dopamine and its respective vehicle.</p

High doses of dopamine reduce sperm motility and acrosomal integrity without affecting the sperm viability over time.

<p><b>A)</b> Effect of dopamine on sperm viability at 1, 3 and 6 hours of incubation. Equine sperm were incubated at 37°C with different concentrations of dopamine (0, 0.01, 0.1 and 1 mM) in Withenn’s buffer and the quantity of viability sperm was calculated using a CASA system, with a minimum of 500 sperm analyzed per experiment. Sperm viability at different times of analysis was normalized to time zero of the control. Results are the mean ± SEM of four independent experiments. <b>B)</b> Effect of dopamine on acrosomal integrity after 1, 3 and 6 hours of incubation. Equine sperm were incubated at 37°C with different dopamine concentrations (0, 0.01, 0.1 and 1 mM) in Withenn’s buffer (enrichment with BSA and bicarbonate) and the percentage of sperms with intact acrosomes was assessed with PSA-FITC staining and by cell count under an epifluorescence microscope. A minimum of 200 sperm were counted. The percentage of intact acrosomes was normalized to time zero of the control without dopamine. Results are the mean ± SEM of four independent experiments. *p<0.05 with respect to the control. <b>C)</b> Effect of dopamine on total sperm motility after incubation for 1, 3 and 6 hours. Equine sperm were incubated at 37°C with different concentrations of dopamine (0, 0.01, 0.1 and 1 mM) in Withenn’s buffer and the quantity of mobile sperm was calculated using a CASA system. A minimum of 500 sperm were analyzed per experiment. Total motility at different times of analysis was normalized to time zero of the control. Results are the mean ± SEM of four independent experiments. **p<0.01 with respect to the control.</p

Dopamine transporter (DAT) is present in equine ejaculated sperm.

<p>The presence of DAT in ejaculated equine sperm was investigated with immunodetection and microscopy methods. <b>A)</b> Sperm were fixed and analyzed by immunofluorescence using an anti-DAT antibody or only secondary antibody as negative control. <b>B)</b> Magnification of DAT immunodetection in sperm. Bar scale is 10 µm. <b>C)</b> 80 µg of total protein extract from rat brain (line 1) and equine sperm (line 2) were analyzed with SDS-PAGE and Western blot using a specific human anti-DAT antibody. Images are representative of 3 independent experiments. <b>D)</b> UREA/SDS-PAGE and Western blot analysis for total protein extract (100 µg) for rat brain (line 1 and 3) and equine sperm protein extract (line 2 and 4), with (line 2 and 4) or without (Line 1 and 3) anti-Dat antibody preabsorbed by immunogenic peptide (SC-7515 P).</p