The autophagy-related protein LC3 is processed in stallion spermatozoa during short-and long-term storage and the related stressful conditions

Use of cooled and frozen semen is becoming increasingly prevalent in the equine industry. However, these procedures cause harmful effects in the sperm cell resulting in reduced cell lifespan and fertility rates. Apoptosis and necrosis-related events are increased during semen cryopreservation. However, a third type of cell death, named autophagy, has not been studied during equine semen storage. Light chain (LC)3 protein is a key component of the autophagy pathway. Under autophagy activation, LC3-I is lipidated and converted to LC3-II. The ratio of LC3-II/LC3-I is widely used as a marker of autophagy activation. The main objective of this study was to investigate whether LC3 is processed during cooling, freezing and the stressful conditions associated with these technologies. A secondary objective was to determine if LC3 processing can be modulated and if that may improve the quality of cryopreserved semen. LC3 processing was studied by Western blot with a specific antibody that recognized both LC3-I and LC3-II. Viability was assessed by flow cytometry. Modulation of LC3-I to LC3-II was studied with known autophagy activators (STF-62247 and rapamycin) or inhibitors (chloroquine and 3-MA) used in somatic cells. The results showed that conversion of LC3-I to LC3-II increased significantly during cooling at 4°C, freezing/thawing and each of the stressful conditions tested (UV radiation, oxidative stress, osmotic stress and changes in temperature). STF-62247 and rapamycin increased the LC3-II/LC3-I ratio and decreased the viability of equine sperm, whereas chloroquine and 3-MA inhibited LC3 processing and maintained the percentage of viable cells after 2 h of incubation at 37°C. Finally, refrigeration at 4°C for 96 h and freezing at −196°C in the presence of chloroquine and 3-MA resulted in higher percentages of viable cells. In conclusion, results showed that an ‘autophagy-like’ mechanism may be involved in the regulation of sperm viability during equine semen cryopreservation. Modulation of autophagy during these reproductive technologies may result in an improvement of semen quality and therefore in higher fertility rates.     

Autophagic proteins regulate cigarette smoke-induced apoptosis: protective role of heme oxygenase-1

Cigarette smoke-induced cell death contributes to the pathogenesis of chronic obstructive pulmonary disease, though the relative roles of apoptosis and autophagy remain unclear. The inducible stress protein heme oxygenase-1 (HO-1) confers cytoprotection against oxidative stress. We examined the relationships between these processes in human bronchial epithelial cells (Beas-2b) exposed to cigarette smoke extract (CSE). CSE induced morphological and biochemical markers of autophagy in Beas-2b cells and induced autophagosome formation as evidenced by formation of GFP-LC3 puncta and electron microscopic analysis. Furthermore, CSE increased the processing of microtubule-associated protein-1 light chain-3 (LC3B-I) to LC3B-II, within 1 hr of exposure. Increased LC3B-II was associated with increased autophagy, since inhibitors of lysosomal proteases and of autophagosome-lysosome fusion further increased LC3B-II levels during CSE exposure. CSE concurrently induced extrinsic apoptosis in Beas-2b cells involving early activation of death-inducing-signaling-complex (DISC) formation and downstream activation of caspases (-8,-9,-3). The induction of extrinsic apoptosis by CSE was dependent in part on autophagic proteins. Reduction of Beclin 1 levels with beclin 1 siRNA inhibited DISC formation and caspase-3/8 activation in response to CSE. LC3B siRNA also inhibited caspase-3/8 activation. The stress protein HO-1 protected against CSE-induced cell death by concurrently downregulating apoptosis and autophagy-related signaling. Adenoviral mediated expression of HO-1 inhibited DISC formation and caspase-3/9 activation in CSE-treated epithelial cells, diminished the expression of Beclin 1, and partially inhibited the processing of LC3B-I to LC3B-II. Conversely, transfection of Beas-2b with ho-1 siRNA augmented CSE-induced DISC formation and increased intracellular reactive oxygen species formation. HO-1 expression augmented CSE-induced phosphorylation of NFkappaB p65 in Beas-2b cells. Consistently, expression of IkappaB, the inhibitor of NFkappaB, increased CSE-induced DISC formation. LC3B siRNA also enhanced p65 phosphorylation. In fibroblasts from beclin 1 heterozygous knockout mice, p65 phosphorylation was dramatically upregulated, while CSE-induced DISC formation was inhibited, consistent with an anti-apoptotic role for NFkappaB and a pro-apoptotic role for Beclin 1. These studies demonstrated an interdependence of autophagic and apoptogenic signaling in CSE-induced cell death, and their coordinated downregulation by HO-1. An understanding of the regulation of cell death pathways during smoke exposure may provide therapeutic strategies in smoke-related illness.     

Autophagic proteins regulate cigarette smoke induced apoptosis: Protective role of heme oxygenase-1

Cigarette smoke-induced cell death contributes to the pathogenesis of chronic obstructive pulmonary disease, though the relative roles of apoptosis and autophagy remain unclear. The inducible stress protein heme oxygenase-1 (HO-1) confers cytoprotection against oxidative stress. We examined the relationships between these processes in human bronchial epithelial cells (Beas-2b) exposed to cigarette smoke extract (CSE). CSE induced morphological and biochemical markers of autophagy in Beas-2b cells. CSE induced autophagosome formation as evidenced by formation of GFP-LC3 puncta and electron microscopic analysis. Furthermore, CSE increased the processing of microtubule-associated protein-1 light chain-3 (LC3B-I) to LC3B-II, within 1 hr of exposure. Increased LC3B-II was associated with increased autophagy, since inhibitors of lysosomal proteases and of autophagosome-lysosome fusion further increased LC3B-II levels during CSE exposure. CSE concurrently induced extrinsic apoptosis in Beas-2b cells involving early activation of death-inducing-signaling-complex (DISC) formation and downstream activation of caspases (-8,-9,-3). The induction of extrinsic apoptosis by CSE was dependent in part on autophagic proteins. Reduction of beclin-1 levels with beclin 1 siRNA inhibited DISC formation and caspase-3/8 activation in response to CSE. LC3B siRNA also inhibited caspase-3/8 activation.     

Storage of red deer epididymides for four days at 5 degrees C: effects on sperm motility,viability, and morphological integrity

The purpose of this study was to assess the sperm motility, the plasma membrane integrity and the morphology of red deer spermatozoa when maintained within epididymides stored for 4 days at 5 degrees C, and to evaluate whether such stored spermatozoa are able to withstand a refrigeration process. Thirty pairs of testes, with attached epididymides, were collected from 30 hunter-killed mature stags (Cervus elaphus hispanicus), and spermatozoa from each one of the pairs were immediately collected in Triladyl medium, evaluated and refrigerated (Control Group). The remaining testes and epididymides were gradually cooled to 5 degrees C and stored for 1, 2, 3, and 4 days (Experimental Groups), after which spermatozoa were processed as described previously for the control group. The effects on spermatozoa that had been stored within epididymides for various times were determined by assaying sperm motility index (SMI), plasma membrane integrity and sperm morphology (SM). In the same way, SMI and SM were assessed after spermatozoa refrigeration at 5 degrees C for 3 hours in different groups (SMI-R, SM-R). There was no significant decrease in plasma membrane integrity of spermatozoa recovered from epididymides stored at 5 degrees C for 4 days. Similarly, the percentage of morphologically normal spermatozoa remained unaffected during the first 3 days of storage. In contrast, during storage sperm motility evaluation revealed significantly (P<0.05) lower SMI values for samples from epididymides stored 2, 3, and 4 days (47.7+/-3.6, 45.5+/-4.4, 44.1+/-5.2) than that of the control group (57.6+/-1.6). Similar results were obtained after refrigeration of spermatozoa in Triladyl at 5 degrees C. These data suggest that it might be possible to recover functional spermatozoa from red deer epididymides stored at 5 degrees C during several days when epididymal spermatozoa cannot be collected and cryopreserved immediately.     

The role of autophagy in human endometrium

Autophagy appears to play an important role in the normal development and maintenance of homeostasis in a variety of tissues, including the female reproductive tract. However, the role of autophagy and the association between autophagy and apoptosis in cyclic remodeling of the human endometrium have not been described. Therefore, we investigated the involvement of autophagy during the human endometrial cycle and its association with apoptosis. Endometrial samples were obtained from 15 premenopausal, nonpregnant women who underwent hysterectomies for benign gynecological reasons. The autophagy-associated protein, microtubule-associated protein 1 light chain 3 alpha (MAP1LC3A), was immunolocalized, and its expression level was measured by Western blot analysis. Apoptosis was evaluated by measuring the expression level of cleaved caspase 3 protein. MAP1LC3A protein was primarily expressed within the endometrial glandular cells and increased during the secretory phase. The expression level of the membrane-bound form of MAP1LC3A (MAP1LC3A-II) also increased as the menstrual cycle progressed, reaching a maximum level during the late secretory phase. This pattern coincided with the expression of cleaved caspase 3. Furthermore, expression of MAP1LC3A-II and cleaved caspase 3 increased in the in vitro-cultured endometrial cancer cells when estrogen and/or progesterone were withdrawn from the culture media to mimic physiological hormonal changes. These findings suggest that endometrial cell autophagy is directly involved in the cyclic remodeling of the human endometrium and is correlated with apoptosis. In addition, we inhibited autophagic processes using 3-methyladenine (3-MA) or bafilomycin A1 (Baf A1) to evaluate the role of autophagy in apoptosis induction in endometrial cancer cells. While the inhibition of autophagosome formation using 3-MA did not decrease apoptosis or cell death, the inhibition of autophagosome degradation by fusion with lysosomes using Baf A1 increased apoptosis and cell death, suggesting that the accumulation of autophagosomes induces apoptosis. Furthermore, Baf A1-induced apoptotic cell death was decreased by the apoptosis inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK). In conclusion, these results indicate that autophagy is involved in the endometrial cell cycle affecting apoptosis and is most prominent during the late secretory phase.     

Increased autophagy in placentas of intrauterine growth-restricted pregnancies

Background

Unexplained intrauterine growth restriction (IUGR) may be a consequence of placental insufficiency; however, its etiology is not fully understood. We surmised that defective placentation in IUGR dysregulates cellular bioenergic homeostasis, leading to increased autophagy in the villous trophoblast. The aims of this work were (1) to compare the differences in autophagy, p53 expression, and apoptosis between placentas of women with normal or IUGR pregnancies; (2) to study the effects of hypoxia and the role of p53 in regulating trophoblast autophagy; and (3) to investigate the relationship between autophagy and apoptosis in hypoxic trophoblasts.

Methodology/Principal Findings

Compared with normal pregnant women, women with IUGR had higher placental levels of autophagy-related proteins LC3B-II, beclin-1, and damage-regulated autophagy modulator (DRAM), with increased p53 and caspase-cleaved cytokeratin 18 (M30). Furthermore, cytotrophoblasts cultured under hypoxia (2% oxygen) in the presence or absence of nutlin-3 (a p53 activity stimulator) had higher levels of LC3B-II, DRAM, and M30 proteins and increased Bax mRNA expression compared with controls cultured under standard conditions. In contrast, administration of pifithrin-α (a p53 activity inhibitor) during hypoxia resulted in protein levels that were similar to those of the control groups. Moreover, cytotrophoblasts transfected with LC3B, beclin-1, or DRAM siRNA had higher levels of M30 compared with the controls under hypoxia. However, transfection with Bcl-2 or Bax siRNA did not cause any significant change in the levels of LC3B-II in hypoxic cytotrophoblasts.

Conclusions/Significance

Together, these results suggest that there is a crosstalk between autophagy and apoptosis in IUGR and that p53 plays a pivotal and complex role in regulating trophoblast cell turnover in response to hypoxic stress.     

Autophagy in Retinal Ganglion Cells in a Rhesus Monkey Chronic Hypertensive Glaucoma Model

Primary open angle glaucoma (POAG) is a neurodegenerative disease characterized by physiological intraocular hypertension that causes damage to the retinal ganglion cells (RGCs). In the past, RGC damage in POAG was suggested to have been attributed to RGC apoptosis. However, in the present study, we applied a model closer to human POAG through the use of a chronic hypertensive glaucoma model in rhesus monkeys to investigate whether another mode of progressive cell death, autophagy, was activated in the glaucomatous retinas. First, in the glaucomatous retinas, the levels of LC3B-II, LC3B-II/LC3B-I and Beclin 1 increased as demonstrated by Western blot analyses, whereas early or initial autophagic vacuoles (AVi) and late or degraded autophagic vacuoles (AVd) accumulated in the ganglion cell layer (GCL) and in the inner plexiform layer (IPL) as determined by transmission electron microscopy (TEM) analysis. Second, lysosome activity and autophagosome-lysosomal fusion increased in the RGCs of the glaucomatous retinas, as demonstrated by Western blotting against lysosome associated membrane protein-1 (LAMP1) and double labeling against LC3B and LAMP1. Third, apoptosis was activated in the glaucomatous eyes with increased levels of caspase-3 and cleaved caspase-3 and an increased number of TUNEL-positive RGCs. Our results suggested that autophagy was activated in RGCs in the chronic hypertensive glaucoma model of rhesus monkeys and that autophagy may have potential as a new target for intervention in glaucoma treatment.     

Vitamin K2 induces autophagy and apoptosis simultaneously in leukemia cells

Vitamin K2 (menaquinone-4: VK2) is a potent inducer for apoptosis in leukemia cells in vitro. HL-60bcl-2 cells, which are derived from a stable transfectant clone of the human bcl-2 gene into the HL-60 leukemia cell line, show 5-fold greater expression of the Bcl-2 protein compared with HL-60neo cells, a control clone transfected with vector alone. VK2 induces apoptosis in HL-60neo cells, whereas HL-60bcl-2 cells are resistant to apoptosis induction by VK2 but show inhibition of cell growth along with an increase of cytoplasmic vacuoles during exposure to VK2. Electron microscopy revealed formation of autophagosomes and autolysosomes in HL-60bcl-2 cells after exposure to VK2. An increase of acid vesicular organelles (AVOs) detected by acridine orange staining for lysosomes as well as conversion of LC3B-I into LC3B-II by immunoblotting and an increased punctuated pattern of cytoplasmic LC3B by fluorescent immunostaining all supported induction of enhanced autophagy in response to VK2 in HL-60bcl-2 cells. However, during shorter exposure to VK2, the formation of autophagosomes was also prominent in HL-60neo cells although nuclear chromatin condensations and nuclear fragments were also observed at the same time. These findings indicated the mixed morphologic features of apoptosis and autophagy. Inhibition of autophagy by either addition of 3-methyladenine, siRNA for Atg7, or Tet-off Atg5 system all resulted in attenuation of VK2-incuded cell death, indicating autophagy-mediated cell death in response to VK2. These data demonstrate that autophagy and apoptosis can be simultaneously induced by VK2. However, autophagy becomes prominent when the cells are protected from rapid apoptotic death by a high expression level of Bcl-2.     

LC3B-II deacetylation by histone deacetylase 6 is involved in serum-starvation-induced autophagic degradation

Autophagy is a conserved mechanism for controlling the degradation of misfolded proteins and damaged organelles in eukaryotes and can be induced by nutrient withdrawal, including serum starvation. Although differential acetylation of autophagy-related proteins has been reported to be involved in autophagic flux, the regulation of acetylated microtubule-associated protein 1 light chain 3 (LC3) is incompletely understood. In this study, we found that the acetylation levels of phosphotidylethanolamine (PE)-conjugated LC3B (LC3B-II), which is a critical component of double-membrane autophagosome, were profoundly decreased in HeLa cells upon autophagy induction by serum starvation. Pretreatment with lysosomal inhibitor chloroquine did not attenuate such deacetylation. Under normal culture medium, we observed increased levels of acetylated LC3B-II in cells treated with tubacin, a specific inhibitor of histone deacetylase 6 (HDAC6). However, tubacin only partially suppressed serum-starvation-induced LC3B-II deacetylation, suggesting that HDAC6 is not the only deacetylase acting on LC3B-II during serum-starvation-induced autophagy. Interestingly, tubacin-induced increase in LC3B-II acetylation was associated with p62/SQSTM1 accumulation upon serum starvation. HDAC6 knockdown did not influence autophagosome formation but resulted in impaired degradation of p62/SQSTM1 during serum starvation. Collectively, our data indicated that LC3B-II deacetylation, which was partly mediated by HDAC6, is involved in autophagic degradation during serum starvation.     

The role of autophagy in corpus luteum regression in the rat

Autophagy is associated with luteal cells death during regression of the corpus luteum (CL) in some species. However, the involvement of autophagy or the association between autophagy and apoptosis in CL regression are largely unknown. Therefore, we investigated the role of autophagy in CL regression and its association with apoptosis. Ovaries were obtained from pseudopregnant rats at Days 2 (early), 7 (mid-), and 14 and 20 (late-luteal stage) of the pseudopregnancy; autophagy-associated protein (microtuble-associated protein light chain 3 [LC3]) was immunolocalized and its expression level was measured. Luteal cell apoptosis was evaluated by measuring cleaved caspase 3 expression. LC3 expression increased slightly from early to mid-luteal stage, with maximal levels detected at the late-luteal stage in steroidogenic luteal cells. The expression level of the membrane form of LC3 (LC3-II) also increased during luteal stage progression, and reached a maximum at the end point of late-luteal stage (Day 20). This pattern coincided with cleaved caspase 3 expression. Furthermore, LC3-II expression increased, as did levels of cleaved caspase 3 in luteal cells cultured with prostaglandin F(2alpha) known to induce CL regression. These findings suggest that luteal cell autophagy is directly involved in CL regression, and is correlated with increased apoptosis. In addition, autophagic processes were inhibited using 3-methyladenine or bafilomycin A1 to evaluate the role of autophagy in apoptosis induction. Inhibition of autophagosome degradation by fusion with lysosomes (bafilomycin A1) increased apoptosis and cell death. Furthermore, inhibition of autophagosome formation (3-methyladenine) decreased apoptosis and cell death, suggesting that the accumulation of autophagosomes induces luteal cell apoptosis. In conclusion, these results indicate that autophagy is involved in rat luteal cell death through apoptosis, and is most prominent during CL regression.     

Morphological and biochemical analysis of cell death in human ejaculated spermatozoa.

We have analyzed the type of cell death occurring in human normal ejaculated spermatozoa. Sperm cells were prepared either by centrifugation alone (group 1) or by density gradient centrifugation (group 2) and were cultured for 24 hrs. Cells were examined after 4 and 24 hrs. By comparison unprepared spermatozoa were used as a control group. Necrosis was investigated by intra-cellular vital stain penetration and electron microscopy. Apoptosis was researched by DAPI staining, annexin V-binding, electron microscopy, DNA fragmentation and PARP cleavage. In group 1, after 4 hrs., there was a mixture of spermatozoa dead either by necrosis or apoptosis while after 24 hrs., necrosis was prominent. Similar findings were observed in the control group. In contrast, in group 2 apoptosis was the major form of cell death of spermatozoa after 24 hrs. of culture. These findings suggest that apoptosis can be an important factor when spermatozoa are used for assisted reproductive technologies.     

Effect of centrifugation on in vitro survival of fresh diluted canine spermatozoa

Prostatic fluid is unsuitable for preserving dog semen at 4 degrees C and exerts harmful effects upon the spermatozoa during the freezing process. Centrifugation immediately after sperm collection is a common method to remove prostatic admixture. In the present study, dog semen, diluted to 25 x 10(6)/ml, was exposed for 5 min to four different centrifugation speeds (180 x g, 720 x g, 1620 x g and 2880 x g) to determine subsequent sperm losses in the supernatant and to assess sperm survival over time. Using 180 x g as centrifugation speed, 8.9% of the sperm cells was lost upon supematant removal. Using 720 x g, 1620 x g or 2880 x g, sperm losses were lower, 2.3, 0.4 and 0.006%, respectively. After centrifugation, the sperm pellet was rediluted in egg-yolk-Tris extender, cooled and stored for 3 days at 4 degrees C. Motility, progressive motility, membrane integrity and sperm morphology were assessed daily. Acrosomal status was assessed after 3 days of storage. The only functional parameter which was influenced by centrifugation speed was membrane integrity as evaluated by means of SYBR14-PI staining: significantly more dead and moribund sperm cells were found after centrifugation at 1620 x g and 2880 x g after 48 and 72 h of storage at 4 degrees C. When higher initial sperm concentrations (50 x 10(6), 75 x 10(6) or 100 x 10(6)/ml) were evaluated for sperm losses, less than 2.3% of the initial total sperm cells was lost at lower centrifugation speeds. We conclude that centrifuging dog sperm for 5 min at 720 x g is the best strategy to remove prostatic fluid because the loss of sperm cells is acceptable and the functional parameters of the spermatozoa are well preserved, even after 3 days of storage.     

Dendrimer-mediated siRNA delivery knocks down Beclin 1 and potentiates NMDA-mediated toxicity in rat cortical neurons

Autophagy is an important process which plays a key role in cellular homeostasis by degrading cytoplasmic components in the lysosomes, which facilitates recycling. Alterations to normal autophagy have been linked to excitotoxicity, but the mechanisms governing its signal transduction remain unclear. The aim of this study was to explore the role of autophagy in neuronal excitotoxic death by delivering small interfering RNA (siRNA) to rat cortical neurons, using a dendrimer to silence the autophagy-related gene 6 (beclin 1) and to determine the role of autophagy in excitotoxicity. We have found that the dendrimer is very efficient to deliver siRNA to rat cortical neurons, leading to almost complete removal of the target protein Beclin 1. In addition, NMDA increases autophagy markers, such as the protein levels of Beclin 1, the microtubule-associated light chain 3 (LC3) B-II/LC3B-I ratio, and monodansylcadaverine (MDC) labeling in rat cortical neurons. Moreover, NMDA also increases the formation of autophagosomes observed under a transmission electron microscope. Silencing beclin 1 expression blocked NMDA-induced autophagy. Moreover, Beclin 1 removal potentiated NMDA-induced neuronal death indicating that autophagy plays a protective role during excitotoxicity and suggesting that targeting autophagy might be a helpful therapeutic strategy in neurodegenerative diseases.     

Immunohistochemical and ultrastructural visualization of different routes of oocyte elimination in adult rats

Cell death is a process for maintaining homeostasis in tissues and organs. In the ovary, apoptotic cell death has been implicated in follicular atresia; in the elimination of the follicles that are not ovulated during adult life. Recent studies indicate that apoptosis and autophagy are two programmed processes of cell death. Apoptosis is performed by proteases called caspases and leads to such morphological traits as DNA fragmentation. Autophagy, in turn, is characterized by the exacerbated formation of autophagosomes; a process in which the amount of the LC3 and Lamp 1 proteins increases. In this study, oocytes from all stages of the estrous cycle of Wistar rats were analyzed. The apoptosis process was identified by immunodetecting active Caspase-3 and locating DNA fragmentation using the TUNEL technique. Autophagy was evaluated through immunodetection of the LC3 and Lamp 1 proteins, and by ultrastructural localization of autophagic vesicle formation. All techniques were conducted using the same oocytes. Results show that all phases of the estrous cycle contain dying oocytes that test positive simultaneously for apoptosis and autophagy markers. The highest level of apoptosis was found during estrus; while the proestrous stage had the highest level of autophagy. The diestrous and metestrous phases were characterized by a high frequency of the presence of markers of apoptosis and autophagy in the same oocyte. Our results demonstrate that during oocyte elimination in adult rats the proteins involved in both processes, apoptosis and autophagy, are present in the same cell at the same time.     

Apoptose au cours de la spermatogenèse et dans le sperme éjaculé

It has become clear in recent years that programmed cell death occurs spontaneously in the cycle of the seminiferous epithelium. Induced germ cell apoptosis occurs at specific stages of the spermatogenic cycle and the existence of supracellular control of germ cell death during spermatogenesis has been documented. If apoptosis is a key phenomenon in the control of sperm production, the existence and role of apoptosis in ejaculated sperm cells remain controversial. Apoptosis — as determined by DNA fragmentation (TUNEL) and ultrastructural analysis — is abnormally frequent in the sperm cells of the ejaculate of sterile men with classical biochemical and ultrastructural pattern. In this review, we discuss the possible origins of DNA damage in ejaculated human spermatozoa and the consequences of DNA damage if the apoptotic spermatozoa is used for ICSI. Percentages of DNA fragmentation in human ejaculated sperm are correlated with fertilization rates both after FIV and ICSI. Detection of DNA fragmentation in human sperm could provide additional information about the biochemical integrity of sperm and may be used in future studies for fertilization failures not explained by conventional sperm parameters. However, the analysis of other molecular markers of apoptosis (Fas, Annexine V ...) is necessary to assess the role of apoptosis in human ejaculated sperm cells.     

Comparative analysis of apoptotic pathways in rat, mouse, and hamster spermatozoa

Apoptosis is a type of cell death characterized by the activation of a family of cysteine-proteases called caspases. We made a comparative study to determine the presence of several caspases and other regulators of apoptosis in rat, mouse, and hamster spermatozoa. Our results showed that the three species have both active and inactive caspases-8 and -3, the proapoptotic protein BID, p53, and the endogenous caspase inhibitor cIAP-1. However, we did not find evidence for the presence of active caspase-9. The acrosome reaction (i.e., the exocytic process of sperm acrosome) and sperm viability were not affected by the presence of a general caspase inhibitor. On the other hand, valinomycin, which promotes caspase-dependent cell death in somatic cells, induced caspase-independent cell death in spermatozoa. TRAIL, a ligand whose receptor induces apoptosis in malignant cells, did not have any effect in the viability of mouse spermatozoa, despise the presence of its receptor in rat and mouse, but not in hamster spermatozoa. Therefore, our results strongly suggest that rodent spermatozoa have some components of the apoptotic pathway. However, the role of caspases in mammalian spermatozoa appears to be unrelated to sperm survival or to the acrosome reaction under physiological conditions.     

Seminal carnitine and acetylcarnitine content and carnitine acetyltransferase activity in young Maremmano stallions

The reproductive characteristics and seminal carnitine and acetylcarnitine content as well as carnitine acetyltransferase activity of young Maremmano stallions (n=25) are reported. The stallions were subjected to semen collection in November and January; in each trial two ejaculates were collected 1h apart. The total motile morphologically normal spermatozoa (TMMNS) and the progressively motile spermatozoa at collection and during storage at +4 degrees C were evaluated. Seminal L-carnitine (LC), acetylcarnitine (AC), pyruvate and lactate were measured using spectrophotometric methods, whereas carnitine acetyltransferase activity was measured by radioenzymatic methods. Since there were no major significant differences in seminal and biochemical characteristics between the November and January trials, data were also pooled for the first and second ejaculates. Significant differences (P<0.001) were observed between the first and second ejaculates for sperm count (0.249+/-0.025 versus 0.133+/-0.014x10(9)/ml), total number spermatozoa by ejaculate (12.81+/-1.23 versus 6.36+/-0.77x10(9)), progressively motile spermatozoa (48.6+/-3.0 versus 52.6+/-3.0%) and TMMNS (3.35+/-0.50 versus 2.02+/-0.37x10(9)). In the raw semen the LC and AC were significantly higher in the first ejaculate than in the second (P<0.001), whereas, pyruvate and pyruvate/lactate ratio were higher in the second ejaculate (P<0.05). Seminal plasma AC and LC concentrations resulted higher in the first ejaculate (P<0.001). The pyruvate/lactate ratio was higher in the second ejaculate (P<0.05). Both raw semen and seminal plasma LC and AC concentrations were positively correlated with spermatozoa concentration (P<0.01); in raw semen AC was also correlated to TMMNS (P<0.01). Lactate levels of raw semen was correlated to progressively motile spermatozoa after storage (P<0.01). In the second ejaculate, significant correlations were also observed among AC/LC ratio in raw semen and progressively motile spermatozoa after 48 and 72h of refrigeration. Furthermore, AC levels were correlated to lactate concentration. The positive correlation between LC, AC and spermatozoa concentration, and between AC and TMMNS indicated carnitine as potential semen quality marker. Moreover, the correlation between AC/LC ratio and progressive spermatozoa motility after refrigeration, suggests that carnitine may contribute towards improving the maintenance of spermatozoa viability during in vitro storage.     

Autophagy Activation Is Involved in 3,4-Methylenedioxymethamphetamine (‘Ecstasy’)—Induced Neurotoxicity in Cultured Cortical Neurons

Autophagic (type II) cell death, characterized by the massive accumulation of autophagic vacuoles in the cytoplasm of cells, has been suggested to play pathogenetic roles in cerebral ischemia, brain trauma, and neurodegenerative disorders. 3,4-Methylenedioxymethamphetamine (MDMA or ecstasy) is an illicit drug causing long-term neurotoxicity in the brain. Apoptotic (type I) and necrotic (type III) cell death have been implicated in MDMA-induced neurotoxicity, while the role of autophagy in MDMA-elicited neurotoxicity has not been investigated. The present study aimed to evaluate the occurrence and contribution of autophagy to neurotoxicity in cultured rat cortical neurons challenged with MDMA. Autophagy activation was monitored by expression of microtubule-associated protein 1 light chain 3 (LC3; an autophagic marker) using immunofluorescence and western blot analysis. Here, we demonstrate that MDMA exposure induced monodansylcadaverine (MDC)- and LC3B-densely stained autophagosome formation and increased conversion of LC3B-I to LC3B-II, coinciding with the neurodegenerative phase of MDMA challenge. Autophagy inhibitor 3-methyladenine (3-MA) pretreatment significantly attenuated MDMA-induced autophagosome accumulation, LC3B-II expression, and ameliorated MDMA-triggered neurite damage and neuronal death. In contrast, enhanced autophagy flux by rapamycin or impaired autophagosome clearance by bafilomycin A1 led to more autophagosome accumulation in neurons and aggravated neurite degeneration, indicating that excessive autophagosome accumulation contributes to MDMA-induced neurotoxicity. Furthermore, MDMA induced phosphorylation of AMP-activated protein kinase (AMPK) and its downstream unc-51-like kinase 1 (ULK1), suggesting the AMPK/ULK1 signaling pathway might be involved in MDMA-induced autophagy activation.     

Polyphyllin VII Induces an Autophagic Cell Death by Activation of the JNK Pathway and Inhibition of PI3K/AKT/mTOR Pathway in HepG2 Cells

Polyphyllin VII (PP7), a pennogenyl saponin isolated from Rhizoma Paridis, exhibited strong anticancer activities in various cancer types. Previous studies found that PP7 induced apoptotic cell death in human hepatoblastoma cancer (HepG2) cells. In the present study, we investigated whether PP7 could induce autophagy and its role in PP7-induced cell death, and elucidated its mechanisms. PP7 induced a robust autophagy in HepG2 cells as demonstrated by the conversion of LC3B-I to LC3B-II, degradation of P62, formation of punctate LC3-positive structures, and autophagic vacuoles tested by western blot analysis or InCell 2000 confocal microscope. Inhibition of autophagy by treating cells with autophagy inhibitor (chloroquine) abolished the cell death caused by PP7, indicating that PP7 induced an autophagic cell death in HepG2 cells. C-Jun N-terminal kinase (JNK) was activated after treatment with PP7 and pretreatment with SP600125, a JNK inhibitor, reversed PP7-induced autophagy and cell death, suggesting that JNK plays a critical role in autophagy caused by PP7. Furthermore, our study demonstrated that PP7 increased the phosphorylation of AMPK and Bcl-2, and inhibited the phosphorylation of PI3K, AKT and mTOR, suggesting their roles in the PP7-induced autophagy. This is the first report that PP7 induces an autophagic cell death in HepG2 cells via inhibition of PI3K/AKT/mTOR, and activation of JNK pathway, which induces phosphorylation of Bcl-2 and dissociation of Beclin-1 from Beclin-1/Bcl-2 complex, leading to induction of autophagy.     

Changes in rat spermatozoa function after cooling,cryopreservation and centrifugation processes

Rat sperm cryopreservation is an effective method of archiving valuable strains for biomedical research and handling of rat spermatozoa is very important for successful cryopreservation. The aim of this study was to evaluate changes in rat sperm function during cryopreservation and centrifugation. Epididymal rat spermatozoa were subjected to cooling and freezing–thawing processes and then motility, plasma membrane integrity (PMI), mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were compared before and after minimum centrifugation force (200×g). Cryopreservation decreased sperm motility, PMI, and MMP (P < 0.05). Basal (without ROS inducer, tert-butyl hydroperoxide [TBHP] treatment) and stimulated ROS (with TBHP treatment) were increased in viable cooled spermatozoa compared to viable fresh spermatozoa (P < 0.01), with equal susceptibility to TBHP among fresh, cooled, and frozen–thawed spermatozoa. Centrifugation decreased motility and PMI of frozen–thawed spermatozoa (P < 0.05). Centrifugation decreased basal ROS of all spermatozoa (P < 0.01), while it led to higher susceptibility to TBHP in viable cooled spermatozoa, showing higher increased fold in ROS and decreased rate in viability by TBHP in viable cooled spermatozoa (P < 0.05). Cooling process was the major step of ROS generation, with loss in sperm motility, PMI, and MMP. Centrifugation affected function of cryopreserved spermatozoa. These data suggest that centrifugation makes rat spermatozoa susceptible to external ROS source, in particular during cooling process. Thus, protection from ROS damage and minimizing centrifugation should be considered during cryopreservation and post-thaw use of cryopreserved epididymal rat spermatozoa.