Intracellular Ca2+ Increases and Connexin 43 Hemichannel Opening Are Necessary but Not Sufficient for Thy-1-Induced Astrocyte Migration

Under pro-inflammatory conditions, astrocytes become reactive and acquire a migratory phenotype. Our results show that hemichannels formed by connexin 43 (Cx43) play an important role in Thy-1-induced astrocyte migration. The neuronal protein Thy-1 binds to αvβ3 integrin in astrocytes, thereby leading to intricate signaling pathways that include calcium (Ca2+) release from intracellular stores, opening of Cx43 hemichannels, release of ATP, activation of P2X7 receptor, and Ca2+ influx. However, because these Thy-1 effects occur exclusively in reactive astrocytes, we wondered whether by elevating calcium levels and promoting hemichannel opening we could prompt non-reactive astrocytes to respond to Thy-1. Cx43 immunoreactivity increased at juxta-membrane sites, where hemichannels (not gap junctions) participate in astrocyte polarization and migration stimulated by Thy-1. Also, intracellular Ca2+ increase, due to ionomycin treatment, induced hemichannel opening, but activated astrocyte migration only partially, and this limitation was overcome by pre-treatment with tumor necrosis factor (TNF) and Thy-1. Finally, αvβ3 integrin formed membrane clusters after TNF stimulation or overexpression of β3 integrin. We suggest that these microclusters are required for cells to respond to Thy-1 stimulation. Therefore, the large increase in intracellular Ca2+ and hemichannel opening induced by ionomycin are required, but not sufficient, to permit Thy-1-induced astrocyte migration. Thus, we suggest that proinflammatory stimuli prompt astrocytes to respond to migratory signals of neuronal cells

Contribution of environmental pollutants to male infertily: A working model of germ cell apoptosis induced by plasticizers

Bisphenol A [2,2-bis(4-hydroxyphenyl)propane] (BPA), 4-nonylphenol (NP) and di(2-ethylhexyl)phthalate (DEHP), and its metabolite mono-2-ethylhexyl phthalate (MEHP) are chemicals found in plastics, which act as endocrine disruptors (EDs) in animals, including human. EDs act like hormones in the endocrine system, and disrupt the physiologic function of endogenous hormones. Most people are exposed to different endocrine disruptors and concern has been raised about their true effect on reproductive organs. In the testis, they seem to preferentially attack developing testis during puberty rather than adult organs. However, the lack of information about the molecular mechanism, and the apparently controversial effect observed in different models has hampered the understanding of their effects on mammalian spermatogenesis. In this review, we critically discuss the available information regarding the effect of BPA, NP and DEHP/ MEHP upon mammalian spermatogenesis, a major target of EDs. Germ cell sloughing, disruption of the blood-testis-barrier and germ cell apoptosis are the most common effects reported in the available literature. We propose a model at the molecular level to explain the effects at the cellular level, mainly focused on germ cell apoptosis

A mechanism of male germ cell apoptosis induced by bisphenol-a and nonylphenol involving ADAM17 and p38 MAPK activation

© 2014 Urriola-Muñoz et al.Germ cell apoptosis regulation is pivotal in order to maintain proper daily sperm production. Several reports have shown that endocrine disruptors such as Bisphenol-A (BPA) and Nonylphenol (NP) induce germ cell apoptosis along with a decrease in sperm production. Given their ubiquitous distribution in plastic products used by humans it is important to clarify their mechanism of action. TACE/ADAM17 is a widely distributed extracellular metalloprotease and participates in the physiological apoptosis of germ cells during spermatogenesis. The aims of this work were: 1) to determine whether BPA and NP induce ADAM17 activation; and 2) to study whether ADAM17 and/or ADAM10 are involved in germ cell apoptosis induced by BPA and NP in the pubertal rat testis. A single dose of BPA or NP (50 mg/kg) induces germ cell apoptosis in 21-day-old male rats, which was prevented by a pharmacological inhibitor of ADAM17, but not by an inhibitor of ADAM10. In vitro, we showed tha

Participation of p38 MAPK in the apoptosis induced by EDC in rat testis.

<p>A) Time course of p38 MAPK phosphorylation in 21-day-old rats treated with 50 mg/kg of BPA. B) Time course of p38 MAPK phosphorylation in 21-day-old rats treated with 50 mg/kg of NP. C) Intra-testicular application of 5 µM PD169316 reduces the phosphorylation of p38 MAPK 1 or 2 h after <i>in vivo</i> treatment with BPA or NP. D) The pharmacological inhibitor of p38 MAPK (PD169316) prevents the increase of TUNEL-positive cells in testes of 21-day-old rats treated with 50 mg/kg of BPA or NP. * p<0.05, n = 3.</p

BPA and NP induce apoptosis in rat testes.

<p>A) Cell cycle analysis by flow cytometry of seminiferous tubule cells isolated from 21 day-old rats after 24 h of a single dose of 10, 50 or 100 mg/kg BPA. The horizontal bar indicates the sub-G1 cell population, which is a parameter of apoptosis. B) There is a significant increase in the percentage of sub-G1 cell population at 50 and 100 mg/kg. C) Quantification of pyknotic cells after the injection of 50 mg/kg of BPA or NP. * p<0.05, n = 3.</p

The ADAM17 inhibitor GW280264X prevents <i>in vivo</i> germ cell apoptosis induced by BPA or NP.

<p>A) Upper graph: Intra-testicular injection of 10 µM GW280264X (GW) prior prevents the increase in the number of TUNEL-positive cells in rat testes induced by 50 mg/kg BPA or NP treatment. Middle graph: Intra-testicular injection of 10 µM GI254023X (GI), an ADAM10 inhibitor, does not prevent the increase in the number of TUNEL-positive cells in rat testes induced by 50 mg/kg BPA or NP treatment. Bottom graph: The application of each inhibitor does not interfere with the normal apoptosis level. Right Panel: Representative TUNEL images from rat testis with the different treatments. B) Treatment with 50 mg/kg BPA or NP induces a significant increase in the cleaved form (89 kDa) of PARP on western blot. The addition of 10 µM GW280264X (GW) prevents the increase of the 89 kDa form in rat testes treated with BPA or NP. * p<0.05, n = 3. Bars = 50 µm.</p

BPA and NP induce the sheddase activity in TM4 cells.

<p>A) Non-transfected TM4 cells do not show any detectable shed of AP activity in the culture medium when incubated with BPA. However, NP induces the increase of AP in non-transfected TM4 cells only at concentrations of 10 and 20 µM (D). B, E) The activity of AP significantly increases in the culture medium of transiently transfected TM4 cell with (AP)-NGR-β1 vector, when they are incubated for 24 h in the presence of 0.01–20 µM BPA (B) or NP (E). (C, F) Time course of AP activity release of transiently transfected TM4 cells with (AP)-NGR-β1 using 0.05 µM BPA (C) or NP (F). G) Transfection of TM4 cells with 10 µg of shRNA (clone 1, sh1 10) induces a robust decrease in the levels of mRNA and (H) protein levels of ADAM17. I, J) Silencing of ADAM17 with 10 µg of shRNA1 (shRNA1 10) in transiently transfected TM4 cells with (AP)-NGR-β1 slightly reduces the basal levels of AP shedding and completely prevents the effect of BPA and NP. * p<0.05, n = 3.</p

A single dose 50 mg/kg of BPA and NP induces a significant increase in active caspase-3 and TUNEL-positive cells in rat testis.

<p>A) BPA and NP treatment induce an increase in the number of active caspase-3. Note that caspase-3 positive cells were mainly spermatocytes (arrows) with BPA, whereas mainly spermatogonia were seen in the case of NP (arroheads). B) TUNEL-positive cells (arrows). * p<0.05, n = 3. Bars 50 µm.</p