In a process called capacitation, mammalian sperm gain the ability to fertilize after residing in the female tract. During capacitation the mouse sperm plasma membrane potential (E m ) hyperpolarizes. However, the mechanisms that regulate sperm E m are not well understood. Here we show that sperm hyperpolarize when external Mammalian sperm are not able to fertilize after ejaculation. They acquire this ability only after residing in the female uterine tract for a finite period of time that varies depending on the species. The molecular, biochemical, and physiological changes that occur in sperm while in the female tract are collectively referred to as capacitation (1). Capacitation is associated with changes in membrane properties, enzyme activities, and motility that prepare the sperm for the acrosome reaction and for penetration of the egg vestments prior to fertilization. The molecular basis of capacitation has been partially defined and includes: the removal of cholesterol from the sperm plasma membrane by cholesterol acceptors such as bovine serum albumin (2, 3), modifications in plasma membrane phospholipids, fluxes of HCO 3 Ϫ (4) and other intracellular ions, and increased tyrosine phosphorylation of proteins (5-7). These events are likely to play a role in the induction of hyperactivated motility and the ability of the sperm to undergo a regulated acrosome reaction (for review see Ref. 8).Bovine and mouse sperm capacitation is also accompanied by a plasma membrane hyperpolarization. E m decreases in mouse sperm from Ϫ38 to Ϫ55 mV (4, 9, 10) and in bovine sperm from Ϫ33 to Ϫ66 mV (9). Because capacitation prepares sperm for the acrosome reaction, the capacitation-associated hyperpolarization may regulate the ability of sperm to generate transient Ca 2ϩ elevations during the acrosome reaction induced by physiological agonists (e.g. zona pellucida) (11). In this respect, low voltage-activated T-type Ca 2ϩ channels have been detected in mouse spermatogenic cells (12, 13), and these channels are also present in mature mouse sperm (14, 15). One unique property of low voltage-activated Ca 2ϩ channels is that they inactivate at the resting E m of sperm prior to capacitation (around Ϫ35 mV) (12,14). Thus, if low voltage-activated Ca 2ϩ channels are involved in the regulation of the acrosome reaction, the capacitation-associated sperm hyperpolarization may be required to remove this inactivation (11,16,17).Although the molecular mechanisms by which the sperm E m hyperpolarizes during capacitation are not clear, there exist several potential candidates. demonstrated with patch clamp techniques that inward rectifying K ϩ channels are expressed in mouse spermatogenic cells and proposed that these channels may contribute to the capacitation-associated sperm membrane hyperpolarization. An increase in sperm K ϩ permeability should lead to an E m hyperpolarization, according to the K ϩ equilibrium potential (18). Alternatively, the sperm plasma membrane may become less permeable to Na ϩ . The relatively depolarized mamma...

Mammalian sperm must undergo a series of physiological changes after leaving the testis to become competent for fertilization. These changes, collectively known as capacitation, occur in the female reproductive tract where the sperm plasma membrane is modified in terms of its components and ionic permeability. Among other events, mouse sperm capacitation leads to an increase in the intracellular Ca(2+) and pH as well as to a hyperpolarization of the membrane potential. It is well known that ion channels play a crucial role in these events, though the molecular identity of the particular channels involved in capacitation is poorly defined. In the present work, we report the identification and potential functional role of K(ATP) channels in mouse spermatogenic cells and sperm. By using whole-cell patch clamp recordings in mouse spermatogenic cells, we found K(+) inwardly rectifying (K(ir)) currents that are sensitive to Ba(2+), glucose and the sulfonylureas (tolbutamide and glibenclamide) that block K(ATP) channels. The presence of these channels was confirmed using inhibitors of the ATP synthesis and K(ATP) channel activators. Furthermore, RT-PCR assays allowed us to detect transcripts for the K(ATP) subunits SUR1, SUR2, K(ir)6.1 and K(ir)6.2 in total RNA from elongated spermatids. In addition, immunoconfocal microscopy revealed the presence of these K(ATP) subunits in mouse spermatogenic cells and sperm. Notably, incubation of sperm with tolbutamide during capacitation abolished hyperpolarization and significantly decreased the percentage of AR in a dose-dependent fashion. Together, our results provide evidence for the presence of K(ATP) channels in mouse spermatogenic cells and sperm and disclose the contribution of these channels to the capacitation-associated hyperpolarization.

Functional evidence indicates that voltage-dependent Ca 2+ (Ca v ) channels participate in sea urchin sperm motility and the acrosome reaction (AR), however, their molecular identity remains unknown. We have identified transcripts for two Ca 2+ channel a1 subunits in sea urchin testis similar in sequence to Ca v 1.2 and Ca v 2.3. Antibodies against rat Ca v 1.2 and Ca v 2.3 channels differentially label proteins in the flagella and acrosome of mature sea urchin sperm. The Ca v channel antagonists nifedipine and nimodipine, which inhibit the AR, diminish the intracellular Ca 2+ elevation induced by a K + -induced depolarization in valinomycin-treated sperm. These findings reveal that Ca v 1.2 and Ca v 2.3 channels could participate in motility and/or the AR in sea urchin sperm.

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Recent reports have demonstrated the role of the G protein-coupled estrogen receptor (GPER1) on the growth and proliferation of breast cancer cells. The coupling of GPER1 to estrogen, tamoxifen or fulvestrant triggers cellular signaling pathways (PI3K and ERK) related to cell proliferation. In an effort to develop new therapeutic strategies against breast cancer, we performed an in silico study to explore the binding pose of a set of designed G15 and G1 analogue compounds, including phenol red. First, we included a carboxyl group instead of the acetyl group from G1 to form amides with several moieties to increase the affinity for GPER1. Then, all the target compounds were submitted to an in silico ADMET study. Then, the ligands were coupled to GPER1 using ligand-based virtual screening to finally achieve molecular dynamics simulations of the best molecule on GPER1, as well as of phenol red, to explore its recognition properties. According to the in silico ADMET and docking studies, the best molecule was named G1-PABA ((3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-carboxylic acid). It was synthesized and assayed in vitro in breast cancer (MCF-7 and MDA-MB-231) and normal (MCF-10A) cell lines. Experimental assays showed that the target compound was able to decrease cell proliferation, showing IC50 values of 15.93 µM, 52.92 µM and 32.45 µM in the MCF-7, MDA-MB-231 and MCF-10A cell lines, respectively, after 72 h of treatment. Interestingly, the target compound showed better IC50 values without phenol red, suggesting that phenol red could interfere with the G1-PABA action at GPER, which is present in MCF-7 cells according to PCR studies and explains the cell proliferation effects. In conclusion, a concentration-dependent inhibition of cell proliferation occurred with G1-PABA in the assayed cell lines and could be due to its action on GPER1.

Background:Aconitum napellus (Acn) is used topically to relieve pain, itching and inflammation, and internally to reduce febrile states, among others. Any circadian time-related consequences of Acn administration are unknown. The objective of this study was to explore the effects of two doses of Acn on body temperature (BT) of mice treated at six different times over 24 hours.Materials and Methods:BALB/c female mice were housed in six chambers (six mice each) with air temperature 24 ± 3°C, humidity 60 ± 4%, and a 12-hours light (L)/12-hours dark cycle, but with L-onset staggered by 4 hours between chambers so that study at one external test time resulted in six test times (02, 06, 10, 14, 18 and 22 hours [h] after light onset). Rectal temperature (RT; in °C) was measured at baseline (B) and 1 hour after oral treatment with placebo (P) or two doses of Acn (6C and 30C, two studies each) in six studies over an 8 day span. The difference in RT for each mouse from the respective B + P timepoint mean RT was computed following each Acn treatment, and data from each of the six studies (original RT and difference from B + P) were analyzed for time-effect by analysis of variance (ANOVA) and for circadian rhythm by 24-hour cosine fitting.Results:A circadian rhythm in RT was found at B and after P (mean: 35.58°C vs. 35.69°C; peak: 15:31 h vs. 15:40 h) and after each Acn dose (30C or 6C). Acn induced hyperthermia and the overall change in BT was rhythmically significant for each dose (mean = +1.95°C vs. +1.70°C), with greatest hyperthermia observed during the L-span for each dose (peak = 08:56 h vs. 05:17 h).Conclusion:Acn administered around the clock induced hyperthermia overall and in a time-dependent manner, with greatest effects during the resting (L) span. Thus, time of day may significantly impact the outcome of Acn and other homeopathic treatments and should be considered in determining optimal dosing and treatment time(s) in order to increase the desired outcome and decrease undesired effects.

Based on theoretical studies, compound DAVP042 was synthesized and evaluated in vitro for its antiproliferative activities on several cancer cell lines (A549-lung, MCF-7-breast, HCT116-colon and U937- lymphoid tissue) in comparison to VPA, as well as for its inhibitory activity on HDAC8 using in vitro models. DAVP042 demonstrated to have antiproliferative activity on all cancer cell lines employed, not only suggesting that this compound should be further studied, but also demonstrating that the methodology herein employed is appropriated to identify new therapeutic candidates.

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