Combined action of X-rays and nonylphenol on mouse sperm

The aim of this study was to assess the effects of 2-weeks’ X-ray and/or nonylphenol (NP) exposure on male mice’s sperm count and quality. Pzh:SFIS mice were exposed to X-rays (0.05 Gy, 0.10 Gy, 0.20 Gy) or to nonylphenol (25 mg/kg bw, 50 mg/kg bw, 100 mg/kg bw) or to both agents (0.05 Gy + 25 mg/kg bw NP, 0.10 Gy + 50 mg/kg bw NP). At 24 h and 5 weeks after the end of exposure the sperm count, morphology and frequency of DNA damage in the male germ cells were estimated. Each agent alone diminished sperm count and morphology. The dose of 0.05 Gy of X-rays decreased the frequency of DNA damage. Combined exposure to lower doses of both agents significantly improved sperm morphology and decreased the level of DNA damage compared to one agent alone. Combined exposure to higher doses reduced the frequency of DNA damage compared to the effect of the appropriate dose of NP. Results of combined exposure to low doses of both agents suggest that 0.05 Gy of X-rays stimulate the DNA damagecontrol system and in consequence repair of DNA caused by X-rays and NP. It may be correlated with increased antioxidant capacity.     

Caspase-independent exposure of aminophospholipids and tyrosine phosphorylation in bicarbonate responsive human sperm cells

Only capacitated sperm cells are able to fertilize egg cells, and this process is triggered by high levels of bicarbonate. Bicarbonate renders the plasma membrane more fluid, which is caused by protein kinase A (PKA)-mediated alterations in the phospholipid (PL) bilayer. We studied exposure of phosphatidylserine (PS) and phosphatidylethanolamine (PE) in human sperm cells. Surface exposure of PS and PE on sperm cell activation in vitro was found to be bicarbonate dependent and restricted to the apical area of the head plasma membrane. The PL scrambling in bicarbonate-triggered human sperm was not related to apoptosis, because the incubated cells did not show any signs of caspases or degeneration of mitochondria or DNA. The PL scramblase (PLSCR) gene family has been implicated in this nonspecific, bidirectional PL movement. A 25-kDa isoform of PLSCR was identified that was homogeneously distributed in human sperm cells. We propose that compartment-dependent activation of PKA is required for the surface exposure of aminophospholipids at the apical plasma membrane of sperm cells. Bicarbonate-induced PL scrambling appears to be an important event in the capacitation process, because the entire intact scrambling sperm subpopulation showed extensive tyrosine phosphorylation, which was absent in the nonscrambling subpopulation. The proportion of live cells with PL scrambling corresponded with that showing capacitation-specific chlortetracyclin staining.     

Biophysical Evaluation of Radiofrequency Electromagnetic Field Effects on Male Reproductive Pattern

There are possible hazardous health effects of exposure to radiofrequency electromagnetic radiations emitted from mobile phone on the human reproductive pattern. It is more effective while keeping mobile phones in pocket or near testicular organs. Present review examines the possible concern on radio frequency radiation interaction and biological effects such as enzyme induction, and toxicological effects, including genotoxicity and carcinogenicity, testicular cancer, and reproductive outcomes. Testicular infertility or testicular cancer due to mobile phone or microwave radiations suggests an increased level of reactive oxygen species (ROS). Though generation of ROS in testis has been responsible for possible toxic effects on physiology of reproduction, the reviews of last few decades have well established that these radiations are very harmful and cause mutagenic changes in reproductive pattern and leads to infertility. The debate will be focused on bio-interaction mechanism between mobile phone and testicular cancer due to ROS formation. This causes the biological damage and leads to several changes like decreased sperm count, enzymatic and hormonal changes, DNA damage, and apoptosis formation. In the present review, physics of mobile phone including future research on various aspects has been discussed.     

Role of cAMP-dependent pathway in eosinophil apoptosis and survival

The survival and apoptosis of eosinophils is of pivotal importance for controlling allergic diseases such as asthma and rhinitis. In this study we have investigated the role for cAMP in regulating eosinophil survival and apoptosis in the absence of eosinophil-active cytokines. The treatment with dibutyryl cyclic AMP (dbcAMP) increased eosinophil survival with a concomitant decrease of apoptosis in a dose-dependent manner. The pretreatment with a protein kinase A (PKA) inhibitor blocked the effects of dbcAMP on survival and apoptosis of eosinophils. The catalytic subunit of PKA was translocated to nucleus in parallel with a robust increase of intracellular cAMP levels upon exposure to dbcAMP but not IL-5, suggesting the separation of PKA activation from the IL-5-induced suppression of eosinophil apoptosis. When eosinophils were treated with pharmacological inhibitors of protein kinases prior to exposure to dbcAMP or IL-5, only the mitogen-activating protein kinase (MAPK) inhibitor, PD098059, was partly able to block dbcAMP-induced augmentation of eosinophil viability, whereas both Janus kinase 2 and MAPK inhibitors effectively interrupted the IL-5-induced prolongation of eosinophil survival. The effects of dbcAMP and these protein kinase inhibitors on eosinophil apoptosis were confirmed by morphologic analysis. We propose that a cAMP-dependent pathway may constitute an important component for regulating eosinophil survival/apoptosisand that cAMP may inhibit eosinophil apoptosis through the activation of PKA and of subsequent MAPK in part.     

The expression and putative role of brain-derived neurotrophic factor and its receptor in bovine sperm

The neurotrophin family of proteins promote the survival and differentiation of nerve cells and are thought to play an important role in development of reproductive tissues. The objective of the present study was to detect the presence of Brain-derived neurotrophic factor (BDNF) and its receptor TrkB in bovine sperm, and explore the potential role of BDNF in sperm function. We demonstrated that both the neorotrophin BDNF and the tyrosine kinase receptor protein TrkB were expressed in ejaculated bovine sperm. Furthermore, BDNF per se was secreted by sperm. Insulin and leptin secretion by bovine sperm were increased (P < 0.01) when cells were exposed to exogenous BDNF, whereas insulin was decreased by K252a. Therefore, we inferred that BDNF could be a regulator of sperm secretion of insulin and leptin through the TrkB receptor. Sperm viability and mitochondrial activity were both decreased (P < 0.05) when the BDNF/TrkB signaling pathway was blocked with K252a. Furthermore, BDNF promoted apoptosis of bovine sperm through TrkB binding (P < 0.05). In conclusion, these observations provided evidence that BDNF secreted by bovine sperm was important in regulation of insulin and leptin secretion in ejaculated bovine sperm. Furthermore, BDNF may affect sperm mitochondrial activity and apoptosis, as well as their viability.     

Deficiency in the c-Jun NH2-terminal kinase signaling pathway confers susceptibility to hyperoxic lung injury in mice

Hyperoxia generates an oxidative stress in the mouse lung, which activates the major stress-inducible kinase pathways, including c-Jun NH2-terminal kinase (JNK). We examined the effect of Jnk1 gene deletion on in vivo responses to hyperoxia in mice. The survival of Jnk1-/- mice was reduced relative to wild-type mice after exposure to continuous hyperoxia. Jnk1-/- mice displayed higher protein concentration in bronchoalveolar lavage (BAL) fluid and increased expression of heme oxygenase-1, a stress-inducible gene, after 65 h of hyperoxia. Contrary to other markers of injury, the leukocyte count in BAL fluid of Jnk1-/- mice was markedly diminished relative to that of wild-type mice. The decrease in BAL leukocyte count was not associated with any decrease in lung myeloperoxidase activity at baseline or after hyperoxia treatment. Pretreatment with inhaled lipopolysaccharide increased BAL neutrophil content and extended hyperoxia survival time to a similar extent in Jnk1-/- and wild-type mice. Associated with increased mortality, Jnk1-/- mice had increased pulmonary epithelial cell apoptosis after exposure to hyperoxia compared with wild-type mice. These results indicate that JNK pathways participate in adaptive responses to hyperoxia in mice.     

Cyclic adenosine 3',5' monophosphate, calcium and protein phosphorylation in flagellar motility

cAMP and calcium are two important regulators of sperm flagellar motility. cAMP stimulates sperm motility by activating cAMP-dependent protein kinase and catalyzing the phosphorylation of sperm proteins. The stimulation of sperm motility by cAMP appears to be at two different levels. Evidence has been presented to suggest that cAMP-dependent phosphorylations may be required in order for motility to be initiated. In addition, cAMP-dependent phosphorylation appears to modulate specific parameters of motility resulting in higher beat frequency or greater wave amplitude. Calcium, on the other hand, when elevated intracellularly to 10(-6) M or higher, inhibits flagellar motility. The calcium-binding protein, calmodulin, appears to mediate a large number of effects of calcium on motility. Evidence suggests that calcium-calmodulin may be involved at the level of the membrane to pump calcium out of the flagellum. In addition, calcium-calmodulin may be involved in the control of axonemal function by regulating dynein ATPase and myosin light chain kinase activities. The identification of cAMP-dependent protein kinase, calmodulin and myosin light chain kinase in the sperm head suggests that cAMP and calcium-dependent phosphorylations are also involved in the control of the fertilization process, i.e., the acrosome reaction, in a manner similar to that known for the control of stimulus/secretion coupling. Finally, the effects of cAMP on flagellar motility are mediated by protein phosphorylation while the effects of calcium on motility are also in part, mediated by effects on protein phosphorylation.     

The efficacy of ultrasound treatment as a reversible male contraceptive in the rhesus monkey

ABSTRACT: BACKGROUND: The use of therapeutic ultrasound as a contraceptive approach has involved nonhuman primates as well as rats and dogs. The current study was undertaken to determine whether this treatment could be a method for reversible contraception, using a model with testes size similar to adult humans. METHODS: Two methods of ultrasound exposure were used, either the transducer probe at the bottom of a cup filled with saline (Cup) or direct application to the surface of the scrotum (Direct). Four adult rhesus (Macaca mulatta) males with normal semen parameters were treated with therapeutic ultrasound at 2.5 W/cm(2) for 30 min. Treatment was given 3 times, one every other day on a Monday-Wednesday-Friday schedule. For each male, semen quality was evaluated a minimum of three times over several months prior to ultrasound exposure and weekly for two months following ultrasound treatment. RESULTS: Semen samples from all males, regardless of exposure method, exhibited a decrease in the percentage of motile sperm following ultrasound treatment. There was an average reduction in motility of 40% the week following treatment. Similarly, curvilinear velocity and the percentage of sperm with a normally shaped flagellum were also reduced in all males following ultrasound treatment. A significant reduction in the total number of sperm in an ejaculate (total sperm count) was only observed in males that received ultrasound via the cup method. Following treatment via the cup method, males exhibited up to a 91.7% decrease in average total sperm count (n = 2). Sperm count did not approach pre-treatment levels until 8 weeks following ultrasound exposure. CONCLUSIONS: The sustained reduction in sperm count, percent motility, normal morphology, and sperm vigor with the cup exposure method provides proof of principle that testicular treatment with ultrasound can be an effective contraceptive approach in humans.     

Les spermatozoïdes immobiles frais ou décongelés en fécondation assistée

Contrasting with sperm count or morphology, complete lack of mobile sperm may seriously impair ICSI fertilization and pergnancy rate. In three cases with flagellar skeleton abnormalities [dynein arm absence] only immobile sperm were found in the ejaculate. Following repeated ejaculations, higher rates of viable spermatozoa and even some motile spermatozoa could be found. Some times, in nonobstructive azoospermia, extensive sperm search didn't allow us to find but immobile sperm mostly, with very few motile sperm cells, not enough for the microinjection of all oocytes. The third group of immobile sperm is iatrogenic, following freezing and thawing surgically retrieved, testicular or epididymal spermatozoa in order to avoid repeated surgical retrieval. Following thawing, one find frequently very few motile spermatozoa that may be not enough for all retrieved oocytes and it might be necessary to inject some eggs with immobile spermatozoa. The outcome of ICSI using mobile and immobile sperm was compared in the three above mentioned groups: 1-immobile ejaculated sperm with flagellar defects, 2-immobile sperm discovered in the ejaculate after extensive sperm search and 3- immobile frozen-thawed testicular or epididymal spermatozoa. The results of ICSI in these groups show that fertilizing ability of fresh or frozenthawed immobile spermatozoa is not significantly different from ICSI with mobile sperm from the same origin. More over, in the first group with flagellar abnormalities, repeated ejaculations allowed us significantly increase sperm viability and fertilization ability. Finding only immobile fresh or frozen-thawed sperm the day of egg retrieval should not lead us to ICSI cancellation. Pregnancies may occur with such immobile sperm.     

Dietary grape seed procyanidin extract protects against lead-induced heart injury in rats involving endoplasmic reticulum stress inhibition and AKT activation

To investigate the protective role of grape seed procyanidin extract (GSPE) against lead-induced heart injury and the possible molecular mechanism associated with this event, Wistar rats were orally given GSPE (200 mg/kg) daily with or without lead acetate (PbA) (0.5 g/L) in drinking water for 56 d. GSPE attenuated oxidative stress, heart dysfunction, and lead accumulation in lead-exposed rat hearts. Meanwhile, GSPE inhibited the protein kinase RNA-like endoplasmic reticulum (ER) kinase/eukaryotic initiation factor 2α signaling pathway, and promoted protein kinase B (AKT) and glycogen synthase kinase 3β phosphorylation altered by lead, and regulated lead-activated apoptosis and its related signaling pathway. This study suggests that dietary GSPE ameliorates lead-induced heart injury associated with ER stress inhibition and AKT activation. Dietary GSPE may be a protector against lead-induced heart injury and a novel therapy for lead exposure.     

Up-regulation of the association between heat shock protein 90 and endothelial nitric oxide synthase prevents high glucose-induced apoptosis in human endothelial cells

Hyperglycemia is the hallmark of diabetes mellitus. Poor glycemic control is correlated with increased cardiovascular morbidity and mortality. High glucose can trigger endothelial cell apoptosis by de-activation of endothelial nitric oxide synthase (eNOS). eNOS was recently demonstrated to be extensively regulated by Akt and heat shock protein 90 (HSP90). Yet, little is known about the molecular mechanisms that regulate eNOS activity during high glucose exposure. The present study was designed to determine the involvement of protein interactions between eNOS and HSP90 in high glucose-induced endothelial cell apoptosis. The protein interaction of eNOS/HSP90 and eNOS/Akt were studied in cultured human umbilical vein endothelial cells (HUVECs) exposed to either control-level (5.5 mM) or high-level (33 mM) glucose for different durations (2, 4, 6, and 24 h). The results showed that the protein interactions between eNOS and HSP90 and between eNOS and Akt and the phosphorylation of eNOS were up-regulated by high glucose exposure for 2-4 h. With longer exposures, these effects decreased gradually. During early hours of exposure, the protein interactions of eNOS/HSP90 and eNOS/Akt and the phosphorylation of eNOS were all inhibited by geldanamycin, an HSP90 inhibitor. High glucose-induced endothelial cell apoptosis was also enhanced by geldanamycin and was reversed by NO donors. LY294002, a phosphatidylinositol 3 (PI3) kinase inhibitor, inhibited the association of eNOS/Akt and the phosphorylation of eNOS but had no effect on the interaction between eNOS and HSP90 during early hours of exposure. From our results we propose that, in HUVECs, during early phase of high glucose exposure, apoptosis can be prevented by enhancement of eNOS activity through augmentation of the protein interaction between eNOS and HSP90 and recruitment of the activated Akt. With longer exposure, dysregulation of eNOS activity would result in apoptosis. The present study provides a molecular basis for the effects of eNOS in the prevention of endothelial cells apoptosis during early phase of high glucose exposure. These observations may contribute to the understanding of the pathogenesis of vascular complications in diabetes mellitus.     

Regulatory and necrotic volume increase in boar spermatozoa

Spermatozoa of many species initially respond to hypotonicity as perfect osmometers. Thereafter they undergo a regulatory process resulting in a decrease in cell volume, similar to that reported for somatic cells. Regulatory volume increase (RVI), a complementary process which is assumed to occur following initial shrinkage of sperm volume after exposure to a hypertonic medium, has not yet been described in detail for spermatozoa. In this study, we investigated whether spermatozoa are able to regulate their volume after hypertonic stress and whether this ability is maintained in preserved sperm. Cell volume changes were recorded using electronic cell sizing. Sperm response to the ion channels blockers quinidine, tamoxifen, and dydeoxyforskolin, and to protein kinase/phosphatase inhibitors lavendustin, staurosporine, and vanadate was studied to investigate possible mechanisms of RVI. Annexin V staining was used in combination with propidium iodide to determine whether hypertonic stress may induce apoptosis. Overall protein tyrosine phosphorylation under hypertonic conditions was measured via flow cytometry using antiphosphotyrosine antibody. Spermatozoa exposed to hypertonic stress initially responded with an abundant subpopulation according to the perfect osmometer model and recovered their volume from this shrinkage after 20 min. RVI was inhibited by quinidine and tamoxifen, which indicates the involvement of the important cellular ions sodium and chloride in this process. Volume regulatory ability was essentially maintained during storage of liquid semen. However, the response of the sperm population was heterogeneous. A second population raised, containing spermatozoa with larger volumes, which demonstrated irregularities in the volume response with respect to osmotic challenge, ion channel blockers, and storage. Under hypertonic conditions, both protein kinase inhibitors (PKI) led to increased isotonic volumes and to elevated initial relative volumes and subsequent volume decrease. RVI was inhibited by the vanadate. Hypertonic stress did not result in an increase in early apoptotic cells, but produced a shift toward late necrotic cells. Substitution of sodium and chloride by choline and sulfate resulted in decreased isotonic volume of sperm treated with lavendustin. Tyrosine phosphorylation levels were reduced after 20 min under hypertonic conditions. It was concluded that RVI is regulated via a protein tyrosine kinase-dependent pathway, and that dephosphorylation occurs when volume regulation is required. The necrotic volume increase (NVI) is associated with the accumulation of sodium and chloride following uncontrolled opening of the channels. The ability to regulate volume after exposure to hypertonic conditions is important for sperm functionality and can have practical applications in spermatological diagnostics and cryopreservation.     

2.45-GHz microwave irradiation adversely affects reproductive function in male mouse,Mus musculus by inducing oxidative and nitrosative stress

Abstract

Electromagnetic radiations are reported to produce long-term and short-term biological effects, which are of great concern to human health due to increasing use of devices emitting EMR especially microwave (MW) radiation in our daily life. In view of the unavoidable use of MW emitting devices (microwaves oven, mobile phones, Wi-Fi, etc.) and their harmful effects on biological system, it was thought worthwhile to investigate the long-term effects of low-level MW irradiation on the reproductive function of male Swiss strain mice and its mechanism of action. Twelve-week-old mice were exposed to non-thermal low-level 2.45-GHz MW radiation (CW for 2 h/day for 30 days, power density = 0.029812 mW/cm² and SAR = 0.018 W/Kg). Sperm count and sperm viability test were done as well as vital organs were processed to study different stress parameters. Plasma was used for testosterone and testis for 3β HSD assay. Immunohistochemistry of 3β HSD and nitric oxide synthase (i-NOS) was also performed in testis. We observed that MW irradiation induced a significant decrease in sperm count and sperm viability along with the decrease in seminiferous tubule diameter and degeneration of seminiferous tubules. Reduction in testicular 3β HSD activity and plasma testosterone levels was also noted in the exposed group of mice. Increased expression of testicular i-NOS was observed in the MW-irradiated group of mice. Further, these adverse reproductive effects suggest that chronic exposure to nonionizing MW radiation may lead to infertility via free radical species-mediated pathway.     

MAPK and mTOR pathways are involved in cadmium-induced neuronal apoptosis

Cadmium (Cd) may be accumulated in human body through long-term exposure to Cd-polluted environment, resulting in neurodegeneration and other diseases. To study the mechanism of Cd-induced neurodegeneration, PC12 and SH-SY5Y cells were exposed to Cd. We observed that Cd-induced apoptosis in the cells in a time- and concentration-dependent manner. Cd rapidly activated the mitogen-activated protein kinases (MAPK) including extracellular signal-regulated kinase 1/2 (Erk1/2), c -Jun N-terminal kinase (JNK) and p38. Inhibition of Erk1/2 and JNK, but not p38, partially protected the cells from Cd-induced apoptosis. Consistently, over-expression of dominant negative c- Jun or down-regulation of Erk1/2, but not p38 MAPK, partially prevented Cd-induced apoptosis. To our surprise, Cd also activated mammalian target of rapamycin (mTOR)-mediated signaling pathways. Treatment with rapamycin, an mTOR inhibitor, blocked Cd-induced phosphorylation of S6K1 and eukaryotic initiation factor 4E binding protein 1, and markedly inhibited Cd-induced apoptosis. Down-regulation of mTOR by RNA interference also in part, rescued cells from Cd-induced death. These findings indicate that activation of the signaling network of MAPK and mTOR is associated with Cd-induced neuronal apoptosis. Our results strongly suggest that inhibitors of MAPK and mTOR may have a potential for prevention of Cd-induced neurodegeneration.     

Adenosine induces endothelial apoptosis by activating protein tyrosine phosphatase: a possible role of p38alpha

Endothelial cell (EC) apoptosis is important in vascular injury, repair, and angiogenesis. Homocysteine and/or adenosine exposure of ECs causes apoptosis. Elevated homocysteine or adenosine occurs in disease states such as homocysteinuria and tissue necrosis, respectively. We examined the intracellular signaling mechanisms involved in this pathway of EC apoptosis. Inhibition of protein tyrosine phosphatase (PTPase) attenuated homocysteine- and/or adenosine-induced apoptosis and completely blocked apoptosis induced by the inhibition of S-adenosylhomocysteine hydrolase with MDL-28842. Consistent with this finding, the tyrosine kinase inhibitor genistein enhanced apoptosis in adenosine-treated ECs. Adenosine significantly elevated the PTPase activity in the ECs. Mitogen-activated protein kinase activities were examined to identify possible downstream targets for the upregulated PTPase(s). Extracellular signal-regulated kinase (ERK) 1 activity was slightly elevated in adenosine-treated ECs, whereas ERK2, c-Jun NH(2)-terminal kinase-1, or p38beta activities differed little. The mitogen-activated protein kinase-1 inhibitor PD-98059 enhanced DNA fragmentation, suggesting that increased ERK1 activity is a result but not a cause of apoptosis in adenosine-treated ECs. Adenosine-treated ECs had diminished p38alpha activity compared with control cells; this effect was blunted on PTPase inhibition. These results indicate that PTPase(s) plays an integral role in the induction of EC apoptosis upon exposure to homocysteine and/or adenosine, possibly by the attenuation of p38alpha activity.     

Sperm count, morphology and fluorescent body frequency in autopsy service workers exposed to formaldehyde

A battery of monitoring tests that could indicate genetic damage was used to investigate occupational formaldehyde exposure in a population of a hospital autopsy service workers. 11 exposed individuals and 11 matched controls were evaluated for sperm count, abnormal sperm morphology and 2F-body frequency. Subjects were matched for sex, age and customary use of alcohol, tobacco and marijuana. Additional information was collected on health, medications and other exposures to toxins. 10 subjects were employed for 4.3 months (range 1-11 months) prior to the first sample and 1 was employed for several years. Formaldehyde exposures were episodic but with a time weighed average between 0.61 and 1.32 ppm (weekly exposure range 3-40 ppm X h). Exposed and control subjects were sampled 3 times at 2-3 month intervals. Sperm morphology was also evaluated in B6C3F1 mice after 5 daily oral doses of 100 mg/kg formalin. No increase in abnormal morphology was detected in the treated animals. In humans, no statistically significant differences were observed between the exposed and control groups for the observed variables. Reduced sperm count correlated with increased abnormal morphology and 2F-body frequency in the exposed group but not in the control group. Evaluation of the impact of incidental exposures suggests a reduced count with marijuana use and increased abnormal morphology with medications used by controls. No effects on sperm were seen from formaldehyde or its metabolites in this population after occupational exposure, nor in mice following a high acute exposure. It is possible that minor effects might have occurred. The lack of an effect in this study may be due to a lack of statistical power to detect effects at this exposure level.     

Calcium/calmodulin and cAMP/protein kinase-A pathways regulate sperm motility in the stallion

In spite of the importance of sperm motility to fertility in the stallion, little is known about the signaling pathways that regulate motility in this species. In other mammals, calcium/calmodulin signaling and the cyclic AMP/protein kinase-A pathway are involved in sperm motility regulation. We hypothesized that these pathways also were involved in the regulation of sperm motility in the stallion. Using immunoblotting, calmodulin and the calmodulin-dependent protein kinase II β were shown to be present in stallion sperm and with indirect immunofluorescence calmodulin was localized to the acrosome and flagellar principal piece. Additionally, inhibition of either calmodulin or protein kinase-A significantly reduced sperm motility without affecting viability. Following inhibition of calmodulin, motility was not restored with agonists of the cyclic AMP/protein kinase-A pathway. These data suggest that calcium/calmodulin and cyclic AMP/protein kinase-A pathways are involved in the regulation of stallion sperm motility. The failure of cyclic AMP/protein kinase-A agonists to restore motility of calmodulin inhibited sperm suggests that both pathways may be required to support normal motility.     

(S)-α-Chlorohydrin Inhibits Protein Tyrosine Phosphorylation through Blocking Cyclic AMP - Protein Kinase A Pathway in Spermatozoa

α-Chlorohydrin is a common contaminant in food. Its (S)-isomer, (S)-α-chlorohydrin (SACH), is known for causing infertility in animals by inhibiting glycolysis of spermatozoa. The aim of present work was to examine the relationship between SACH and protein tyrosine phosphorylation (PTP), which plays a critical role in regulating mammalian sperm capacitation. In vitro exposure of SACH 50 μM to isolated rat epididymal sperm inhibited PTP. Sperm-specific glyceraldehyde 3-phosphate dehydrogenase (GAPDS) activities, the intracellular adenosine 5'-triphosphate (ATP) levels, 3'-5'-cyclic adenosine monophosphate (cAMP) levels and phosphorylation of protein kinase A (PKA) substrates in rat sperm were diminished dramatically, indicating that both glycolysis and the cAMP/PKA signaling pathway were impaired by SACH. The inhibition of both PTP and phosphorylation of PKA substrates by SACH could be restored by addition of cAMP analog dibutyryl-cAMP (dbcAMP) and phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Moreover, addition of glycerol protected glycolysis, ATP levels, phosphorylation of PKA substrates and PTP against the influence of SACH. These results suggested SACH inhibited PTP through blocking cAMP/PKA pathway in sperm, and PTP inhibition may play a role in infertility associated with SACH.     

The phosphorylation of a putative sperm microtubule-associated protein 2 (MAP2) is uniquely sensitive to regulation.

We have identified a bovine sperm phosphoprotein, pp255 (Mr = 255,000), which reacts strongly and specifically with an antibody to rat brain microtubule-associated protein 2 (MAP2). The phosphorylation state of this putative sperm MAP2 in intact bovine epididymal sperm is uniquely sensitive to regulation by intracellular pH (pHi), calcium, isobutyl-3-methylxanthine (MIX), H-8, and fluoride. Increasing pHi by approximately 0.4 units or exposure to calcium (0.1 microM with the ionophore A23187) or to the protein kinase inhibitor, H-8, decreases sperm MAP2 phosphorylation. Decreasing sperm pHi or exposure to MIX or fluoride increases MAP2 phosphorylation. Numerous other detectable sperm phosphoproteins are either unresponsive to most of these modulators or are considerably less sensitive to them. This phosphoprotein co-sediments with the particulate sperm heads during subcellular fractionation, and is not detectable in other sperm fractions. Two-dimensional electrophoresis separates sperm MAP2 into multiple species, indicative of varying degrees of phosphorylation. Sperm MAP2 is phosphorylated on serine residues, changes electrophoretic mobility slightly on one-dimensional gels with changes in phosphorylation levels, and exhibits the highest specific radioactivity of any sperm phosphoprotein observed. The phosphorylation state of sperm MAP2 can be uncoupled from sperm motility levels under several conditions. The co-localization of sperm MAP2 with the head fraction and the unique sensitivity of its phosphorylation level to modulators, which are known to regulate capacitation and the acrosome reaction, suggest that sperm MAP2 phosphorylation may be an intermediate step in the regulation of one or both of these sperm processes.     

Haloperidol-induced neuronal apoptosis: role of p38 and c-Jun-NH(2)-terminal protein kinase

We examined patterns and mechanisms of cell death induced by haloperidol. Cortical cell cultures exposed to 10-100 microM: haloperidol for 24 h underwent neuronal death without injuring glia. The degenerating neurons showed hallmarks of apoptosis, featuring cell body shrinkage, nuclear chromatin condensation and aggregation, nuclear membrane disintegration with intact plasma membrane, and prominent internucleosomal DNA fragmentation. Neither glutamate antagonists nor antioxidants prevented the haloperidol-induced neuronal apoptosis. The c-Jun-NH(2)-terminal protein kinase and p38 mitogen-activated protein kinase were activated within 1 h and were sustained over the next 3 h following exposure of cortical neurons to 30 microM haloperidol. Haloperidol-induced neuronal apoptosis was partially attenuated by 10-30 microM PD169316, a selective inhibitor of p38 mitogen-activated protein kinase. Inclusion of 1 microg/ml cycloheximide, a protein synthesis inhibitor, or 100 ng/ml insulin prevented activation of both kinases and subsequent neuronal death. The present study demonstrates that cortical neurons exposed to haloperidol undergo apoptosis depending on activation of p38 mitogen-activated protein kinase and c-Jun-NH(2)-terminal protein kinase sensitive to cycloheximide and insulin.