Abstract Graphene, an allotrope of carbon in 2D structure, has revolutionised research, development and application in various disciplines since its successful isolation 16 years ago. The single layer of sp2-hybridised carbon atoms brings with it a string of unrivalled characteristics at a fraction of the price of its competitors, including platinum, gold and silver. More recently, there has been a growing trend in the application of graphene in catalysis, either as metal-free catalysts, composite catalysts or as catalyst supports. The unique and extraordinary properties of graphene have rendered it useful in increasing the reactivity and selectivity of some reactions. Owing to its large surface area, outstanding adsorptivity and high compatibility with various functional groups, graphene is able to provide a whole new level of possibilities and flexibilities to design and synthesise fit-for-purpose graphene-based catalysts for specific applications. This review is focussed on the progress, mechanisms and challenges of graphene application in four main reactions, i.e., oxygen reduction reaction, water splitting, water treatment and Fischer–Tropsch synthesis. This review also summarises the advantages and drawbacks of graphene over other commonly used catalysts. Given the inherent nature of graphene, coupled with its recent accelerated advancement in the synthesis and modification processes, it is anticipated that the application of graphene in catalysis will grow exponentially from its current stage of infancy.

In keeping up with the current push for multiliteracies in ELT, the secondary school EL curriculum of Singapore has placed an emphasis on multiliteracies. Students are encouraged to engage with ‘rich language’ through a range of semiotic resources, including the use of multimodal texts. Drawing on the framework of multiliteracies pedagogy that integrates the four components of situated practice, overt instruction, critical framing and transformed practice (New London Group, 1996), this article examines multimodal meaning-making in the enacted EL curriculum in Singapore’s multilingual classrooms. By looking at how visual literacy is taught and how multimodal texts are used in curriculum implementation and pedagogy, I hope to demonstrate that the framework of multiliteracies pedagogy adopted or adapted, has provided a rich environment for students to create engaging and interactive learning opportunities for themselves. I also hope to showcase how visualization training to develop the mind’s eye through carefully designed language learning tasks can enhance students’ visual literacy in an increasingly multi-modal, multi-dimensional world where they are surrounded by an array of semiotic resources across language and culture.

Bisphenol A (BPA), an endocrine-disrupting chemical, has received tremendous attention in the past few decades because of its detrimental health effects. Growing evidence supports that BPA is capable to alter the reproductive performance of the exposed individual. In spermatozoa, it has been reported that BPA increased oxidative stress by the overproduction of reactive oxygen species (ROS), subsequently affects the sperm function, biochemical properties, and fertility. Since antioxidants minimize cellular oxidative stress, therefore may have protective effects against BPA-induced stress. In the present study, we incubated mice spermatozoa for 6 h in a condition that support in vitro fertilization. The sperm incubation media was additionally supplemented with either BPA or BPA together with antioxidants, such as glutathione, vitamin C, and vitamin E. Our results showed that antioxidant significantly decreased the production of ROS that subsequently supports motility and acrosomal integrity of BPA-exposed spermatozoa. Particularly, glutathione and vitamin E inhibit protein kinase-A dependent phosphorylation of sperm proteins subsequently prevented precocious acrosome reaction. In addition, both antioxidants were found to restore fertilization and early embryo development potentiality of BPA-exposed spermatozoa. Therefore, we conclude that antioxidants minimize oxidative stress in spermatozoa in a BPA containing micro-environment, thus avoiding BPA-mediated harmful consequences. The current finding has both theoretical and clinical significance for developing potential remedies of the BPA toxicity.

The ability of conventional semen analysis to predict male fertility is questionable. Since the prediction of male fertility is extremely of importance for the artificial insemination and profitable farm managements in animals, the development of highly sensitive biomarker of male fertility is a prime concern. Porcine Seminal Protein I (PSP-I) and Porcine Seminal Protein II (PSP-II) have been known that they are related with motility, and viability of spermatozoa. Thus, we investigated PSP-I and PSP-II level in boar spermatozoa to predict boar’s fertility. The expressions of PSP-I and PSP-II in spermatozoa from 21 individual boars with different fertility and litter size (litter size ranges from 10.3 – 14.2) were examined using qRT-PCR. Litter size was determined in 530 saws after artificial insemination (AI). In addition, sperm motility, motion kinematics, and capacitation status were measured using computer-assisted sperm analysis and Hoechst 33258/chlortetracycline fluorescence staining, respectively. PSP-I and PSP-II showed significantly negative correlation with litter size (r=0.578; P=0.006 and r=0.456; P=0.038, respectively). Furthermore, receiver-operating curves (ROC) was used to determine the accuracy for the prediction of boar fertility. Therefore we divided into 2 groups based on the median value of litter size. When selecting higher litter size group, PSP-I can predict litter size with overall accuracy 90.48% (sensitivity 88.89, specificity 91.67, negative predictive value 91.67, and positive predictive value 88.89) and PSP-II can predict with overall accuracy 81.82% (sensitivity 55.56, specificity 100.00, negative predictive value 76.47, and positive predictive value 100.00). Interestingly, PSP-I and PSP-II were found to increase 0.76 pups than average litter size (average 12.48) in tested boars. To best of our knowledge, this study is the first trial to investigate the correlation between PSP-I, PSP-II, and litter size. Therefore, we suggest that PSP-I and PSP-II could be considered as promising biomarkers for predicting male fertility and litter size outcome in field condition.

Sperm cryopreservation is well known as a valuable method to preserve the genetic traits. Although many studies have established semen cryopreservation protocols, lack of studies were conducted to discover the differences of sperm proteome and functions between ejaculated and epididymal spermatozoa following to cryopreservation. Therefore, the objective of this study was to (i) evaluate the effect of cryopreservation on bull epididymal spermatozoa and (ii) discover the potential biomarkers, which have highly tolerance to freezing on bull epididymal spermatozoa. Our preliminary study demonstrated that spermatozoa from each bulls have different resistance on freezing during cryopreservation. We divided spermatozoa into two groups according to sperm motility following to cryopreservation; high freezing-tolerant spermatozoa (HFS) and low freezing-tolerant spermatozoa (LFS). Several sperm functional parameters, i.e. sperm motility/motion kinematics, speed parameters, viability, mitochondrial membrane potential, and capacitation status. Our results showed that all parameters except for motion kinematics and capacitation status had significant differences between HFS and LFS. Subsequently, two dimensional electrophoresis were conducted to compare the expression levels of sperm proteome between both groups. Three proteins {glutathione s-transferase mu 5 (GSTM5), voltage-dependent anion-selective channel protein 2 (VDAC2), and ATP stynthase subunit beta (ATP1B1)} were differentially expressed. Based on these results, we propose that epididymal spermatozoa from individual bull have different freezability upon cryopreservation and three differentially expressed proteins might be selected as a biomarker to predict high freezing-tolerant epididymal spermatozoa.

Sperm cryopreservation preserves genetic resources for animal breeding and for human patients who suffers from permanent testicular damage. Although the sperm cryopreservation has been used for many years, the addition of cryoprotective agent (CPA) during cryopreservation negatively affects sperm function and quality. Our previous study reported that the addition of CPA reduced bull sperm physiological functions. However, the sperm cells collected from individual bulls presented different sensitivity to the damage induced by CPA. In the present study, we examined if CPA affect sperm cells acquired from individual bulls. Individual bull spermatozoa were divided into two groups based on motility parameters; high CPA-tolerant sperm (HCS) and low CPA-tolerant sperm (LCS). Our results showed that the HCS group presented good physiological function after CPA exposure, whereas the LCS group showed a significant decrease in the sperm function. We also found differentially expressed five proteins between the HCS and LCS groups, which refer to cytosolic 5′-nucleotidase 1B (NT5C1B), fumarate hydratase (FH), F-actin-capping protein subunit beta (CAPZB), voltage-dependent anion-selective channel protein 2 (VDAC2), and cytochrome b-c1 complex subunit 1 (UQCRC1). NT5C1B and FH showed abundant expression in the HCS group, while the expression of CAPZB, VDAC2, and UQCRC1 was relatively lower in the HCS group than in the LCS group. The current results suggest that NT5C1B, FH, CAPZB, VDAC2, and UQCRC1 can be used as potential markers to predict CPA-tolerable spermatozoa. Those markers provide a reliable tool to select animals and breeds with CPA tolerance.

Bisphenol‒A (BPA) is a known endocrine‒disrupting chemical used extensively to manufacture plastic bottles, canned food linings, thermal receipts, and other commonly used items. BPA is capable of inducing chromosomal alterations in germ cell line, thereby produced transgenerational effects on brain function, social recognition, reproductive diseases, sperm quality, gene expression, and obesity. Here, we aimed to investigate the transgenerational effects of BPA on murine male fertility. Six-week-old male mice (F0) were gavaged with corn oil (control), two different doses of BPA (5 mg, and 50 mg·kg bw-1·day-1),andethinylestradiol(EE,0.4mg·kg bw-1·day-1), dailyfor6weeks. Treated male mice were mated with wild‒type female and sibling pairs were bred up to the third generation (F3) in a similar manner with no further BPA exposure. Testes and spermatozoa were collected from 14-week-old males of all generation (F0 to F3) to evaluate testis weight, sperm function, and fertility. We found that high concentration of BPA significantly increased testicular weight in F2. Although the sperm viability, capacitation status, and intracellular ROS levels were not affected by BPA, however, sperm count, motility, hyperactivated motility, and intracellular ATP levels were significantly altered by BPA, dose dependently. In majority of the cases the effects were prominent in F2 followed by F1 and F0, whereas the effects were diminished in F3 generation. Simultaneously, high concentration of BPA significantly decreased cleavage and blastocyst formation rate in both F1 and F2. Similar inhibitory effects on cleavage and blastocyst were also noted in F1 by low dose of BPA. Depending on these findings we conclude that BPA decreases the fertility potential of exposed males and has an adverse impact on sperm function and fertility in subsequent generations.

Processes of cryopreservation consists of three steps: dilution with the extender/cooling (Step 1), addition of cryoprotectant (Step 2), and freezing/thawing (Step 3) and spermatozoa are exposed different kind of environment and stress in each step. We categorized sperm samples as good freezablitiy (GF), damaged by cryoprotectant (DCP), and damaged by freezing (DF) and identified characteristics of each group in different step of cryopreservation. In Step 2, DCP was significantly decreased in motility, rapid speed and increased in slow speed. DF was significantly decreased in only motility whereas there were no significant difference between GF and DF and significantly higher than DCP in Step 2. Motility, rapid, medium speed of all group were significantly decreased in Step 3 and GF was significantly higer than other groups. AR pattern of all groups were significantly increased in Step 3 whereas GF was significantly lower than other groups. Additionally AR pattern of DF was significantly increased in Step 2. F pattern of DF and DCP were significantly decreased in Step 3. There no difference of B pattern in whole process. Mitochondrial activity of DCP was significantly decreased in Step 2 and mtichondrial activity of all groups were significantly decreased in Step 3. However mtichondrial activity of GF was higher than other groups. Viability result shows same significant difference with mitochondrial pattern. The present study compared with various sperm parameters in different groups which has different freezability. We defined different two types of group that damaged from different step of cryopreservation. DF and DCP is mainly damaged in Step 3 and Step 2 respectively. The results of current study suggest that various sperm parameters can be used as physical markers in freezability.

Iron is required for cell viability but is toxic in excess. While the iron-mediated malfunction of testicular cells is well appreciated, the underlying mechanism(s) of this effect and its relationship with fertility are poorly understood. Ferritin is a ubiquitous intracellular protein that controls iron storage, ferroxidase activity, immune response, and stress response in cells. Ferritin light chain protein (FTL) is the light subunit of the Ferritin. Previously, we had identified the FTL in bovine spermatozoa following capacitation. In present study, to investigate the role of Ferritin in sperm function, mice spermatozoa were incubated with multiple doses (1, 10 and 100 μM) of sodium nitroprusside (SNP), an iron donor. SNP was increased Ferritin levels in a dose-dependent manner. The Ferritin was detected on the acrosome in spermatozoa by immunocytochemistry. Short-term exposure of spermatozoa to SNP increased tyrosine phosphorylation and the acrosome reaction (AR). Finally, SNP affected a significant decrease in the rate of fertilization as well as blastocyst formation during early embryonic development. On the basis of these results, we propose that the effects of Ferritin on the AR may reduce overall sperm function leads to poor fertility in males and compromised embryonic development.

Sperm capacitation refers to polymerization of filamentous (F)-actin from globular (G)-actin. While the role of ac-tin-related protein 2/3 (Arp2/3) complex in actin polymerization is well appreciated, the underlying mechanism(s) and its relationship with capacitation are poorly understood. Therefore, to evaluate the potential role of Arp2/3 complex on capacitation, bovine spermatozoa were incubated with multiple doses (1, 10 and 100 μM) of CK-636, an inhibitor of Arp2/3 complex with heparin. The cellular localization of the Arp2/3 complex in spermatozoa was identified by immunohistochemistry, whereas western blot was also applied to detect the protein tyrosine phosphorylation of sperm proteins. Additionally, sperm motility and kinematic parameters were evaluated using a computer-assisted sperm analysis system. CK-636 resulted in significant changes in the ratio of Arp2/3 complex localization between acrosome and equatorial region of the spermatozoa. Short-term exposure of spermatozoa to 100 μM of CK-636 significantly decreased sperm motility, however a non-detectable effect on protein tyrosine phosphorylation was observed during capacitation. On the basis of these results, we propose that Arp2/3 complex is associated with morphological changes during capacitation and compromised sperm motility.

The prediction of male fertility is of paramount importance for breeding animal herds when artificial insemination is applied. While the male fertility assays provide valuable quantitative data, they yield limited information concerning the functional competence of the spermatozoa. The objective of this study was to standardize a method for predicting in vivo fertility in bulls using the capacitation status that was assessed by chlortetracycline (CTC) staining. To optimize the capacitation process, sperm were treated with various concentrations of heparin (0, 10, 20, 50, and 100 μg/mL) and incubated for 10, 20, and 30 min each at 39℃ in 5% CO2. We found that maximum capacitation condition obtained from 10 μg /mL heparin treated sperm cells for 20 min (p<0.05). Optimized methods were used to determine the fertility of 17 batches of frozen bull semen representing a wide range of field fertility levels as indicated by non-return rates (NRR) (35.29% 93.18%). There was no significant correlation between NRR and the percentage of capacitated spermatozoa (B type) and non-capacitated spermatozoa (F type). However, acrosome reacted spermatozoa (AR type) was significantly correlated with NRR (p<0.01). To determine the normal range for the AR type, lower limits of the AR (%) were established as 23% for low fertility (NRR < 75%) using receiver operating characteristic curve. The overall accuracy of the assay was 88.24% for low fertility, sensitivity and specificity were 81.82 and 100%, respectively. These results indicate that capacitation status as measure by CTC staining is a useful predictor of male fertility. Therefore, low and high fertility bulls can be identified primarily by the functional capacitation status.

The objective of this study was to examine the effect of various discontinuous Percoll washing conditions on sperm capacitation status and sperm survival. Frozen epididymal sperm samples from 3 bulls (0.5 ml plastic straws, 6% glycerol in egg yolk- Tris-glycerol extender) were thawed in 37℃ water bath for 1 min. To rule out individual variation, 3 sperm samples were mixed after thawing. The mixed samples then were randomly allocated to 12 treatment groups. Briefly, the spermatozoa were centrifuged for three different time lengths (10, 20, and 30 min) at two gravities (300 X g and 700 X g) through two concentrations of discontinuous Percoll density gradient of 1 ml 90%: 1 ml 45% Percoll and 2 ml 90%: 2 ml 45% Percoll to remove extender, debris, and dead spermatozoa. Sperm capacitation status and sperm survival were evaluated using combined Hoechst 33258 and chlortertracycline fluorescence staining assay. The acrosome reacted spermatozoa (AR pattern), uncapaciated spermatozoa (F pattern) and sperm survival were significantly correlated with centrifugation time (p< 0.01). Significantly decreased F pattern observed as centrifugal time increased. As centrifugal time increased, spermatozoa with F pattern decreased and spermatozoa showing AR pattern increased. Moreover, the dead spermatozoa were significantly stimulated in time-dependent manner. However, there were no significant differences in various force of centrifugation and Percoll volume. These results suggest that only centrifugation time significantly affects sperm capacitation status and sperm survival.

Voltage-dependent anion channel (VDAC) is mitochondrial protein of all eukaryotes. It has been reported that VDAC is a large voltage-dependent channel, regulation of ion (including Ca2+), and transportation of various metabolites. Ca2+ is an important factor in sperm function. In our previous study, we found high frequency of VDAC2 expression in spermatozoa from low-fertility bulls. However, to date, there is limited information available on its effects on male fertility. Therefore this experiment was designed to evaluate the effects of VDAC and Ca2+ on sperm function in vitro. To achieve this, four treatment conditions were established with or without Ca2+ and VDAC inhibitor, namely, 4’-diisothiocyano-2,2’-disulfonic acid stilbene (DIDS). Spermatozoa from adult ICR were collected and released into modified Tyrode’s salt media. And then, they were incubated in the different media with or without Ca2+and DIDS for 90 min at 37℃ in 5% CO2. Intracellular pH ([pH]i) and Ca2+ ([Ca2+]i) were measured by their fluorescent indicators, 2,7-bicarboxyethyl-5,6-carboxy- fluorescein acetoxymethyl ester (BCECF- AM) and fura-2 AM, respectively. Western blot of extracted sperm proteins with an anti-phosphotyrosine antibody (pY20) was carried out to determine tyrosine phosphorylation after sperm incubation in different treatments. To evaluate the fertilizing ability after treatments, in vitro fertilization was performed. DIDS significantly decreased [Ca2+]i regardless of Ca2+. [pH]i was efficiently affected by the presence of Ca2+ and/or DIDS. However, the highest decrease of pH level was observed under the presence of DIDS and the absence of Ca2+ in culture condition. Tyrosine phosphorylated protein 1 was significantly different under all treatments. However, tyrosine phosphorylated protein 2 was not significantly different under the presence of DIDS. Fertilization rate was significantly decreased under the presence of DIDS. Blastocyst formation was significantly altered different to compare to control and each treatment group. Therefore it suggests that a voltage-dependent anion channel may involved paramount importance in regulation of male fertility.

Phosphorylation of proteins is a post-translational modification process which plays a significant role in a wide range of cellular processes. Addition or removal of phosphate groups result in conformational changes in proteins leading either to their activation or inactivation. Tyrosine phosphorylation of protein is associated with sperm function in several mammalian species. The control of this process may via the changes in cyclic adenosine monophosphate (cAMP); the changes in cAMP levels that occur in the spermatozoa regulate protein kinase A (PKA) activity which, in turn, leads to the tyrosine phosphorylation of protein substrates by either the activation of sperm tyrosine kinases and/or the inhibition of phosphoprotein phosphatases. Cyclic nucleotides, in particular, cAMP, are important regulators of various maturation events in sperm including capacitation and motility. Interestingly, some environmental chemicals (ECs) may exert broader endocrine disrupting effects through possible modulation of cAMP/PKA second messenger systems. Otherwise, because the mature spermatozoa are transcriptionally inactive, therefore the study of sperm proteins phosphorylation may permit more information about the agents and conditions affects on sperm function. In the present study, to examine the effect of ECs on human sperm function, human spermatozoa were incubated with a group of ECs represent a widespread chemicals in the environment bisphenol A (BPA, 100 μM), nonylphenol (NP, 10 μg/ml), 2,3,7,8-Tetrachlorodibenzo- pdioxin (TCDD, 2.5 μg/ml), genistein (Gen, 100 μM), and the following pesticides, dibromochloropropane (DBCP, 10 μg/ml), atrazine (Atraz, 500 μM), and diazinone (Diaz, 500 μM) for 6 hr at 37℃ in 5% CO2. Then, western blot analysis was carried out using extracted sperm proteins. Antiphosphorylation antibody (pY20) was used to determine sperm tyrosine phosphorylation after EDs treatment. The pY20 antibody labeled three common bands of approximately 90, 110, and 150 KDa. There were no significant differences between negative and positive control groups in regard to the tyrosine phosphorylated proteins except at the band with molecular weight 110 KDa. However, except Diaz treatment group, the other treatment groups showed decreasing (TCDD, Gen, NP, BPA, and DBCP) or increasing (Atraz) in the tyrosine phosphorylated proteins at least in one band from the three common bands studied. Therefore, it sug-gests that ECs effectively alters human sperm function and this effect may detect via their effect on tyrosine phosphorylation pattern.

In mammals, the meiosis division in testes produces equal numbers of two different types of gametes: X chromosome-bearing sperm (X-spermatozoa) and Y chromosomebearing sperm (Y-spermatozoa), which have equal potential to fertilize the oocytes. Therefore, the expected 1: 1 sex ratio is observed. However, under some conditions like endocrine disruptors (EDs) exposure the sex ratio is deviated than the expected with more males or more females. And recently many hypotheses have been postulated to explain the mechanism of sex ratio deviation; however none of them introduced a proven experimental explanation. To solve this enigma, we hypothesized that the differences between X- and Y-spermatozoa survivability under specific conditions due to differences in their chromosome contents are the key leading to the sex ratio alteration. To examine our hypothesis, we combined two techniques; first, hypo-osmotic swelling (HOS) test that was applied to test viability of spermatozoa and second, fluorescence in situ hybridization that was applied on HOS-treated spermatozoa to define sex chromosome composition. In the present study, human spermatozoa were incubated with a group of EDs represent a widespread chemicals in the environment bisphenol A (BPA, 100 μM), nonylphenol (NP, 10 μg/ml), 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, 2.5 μg/ml), genistein (Gen, 100 μM), and the following pesticides, dibromochloropropane (DBCP, 10 μg/ml), atrazine (Atraz, 500 μM), and diazinone (Diaz, 500 μM) for 6 hr at 37℃ in 5% CO2. Then, the viability of spermatozoa and their sex chromosome contents were evaluated simultaneously. Among seven chemicals studied only four chemicals (Atraz, DBCP, TCDD, and Diaz) significantly decreased Y-sperm viability when compared to those of X-spermatozoa in the same treatment group and viability of Y-spermatozoa when compared to those in the negative and positive (DMSO) control groups (p<0.05). Also, in these four treatment groups the sex ratio of live sperm population was significantly lowered compared to the control groups (p<0.05). Otherwise, Gen, BPA, and NP did not show any significant effect on viability of Yspermatozoa or decreasing sex ratio in live sperm population as compared to the control groups. It has been proven that TCDD, DBCP, and the pesticides decrease the sex ratio, but the same effect was not observed in case of Gen, BPA, and NP. From the present findings, there is no doubt that the EDs may alter sex ratio via decreasing Y-spermatozoa viability.

In the last few decades with the industrial revolution many environmental contaminants have estrogenic activity (endocrine disruptors, EDs) are released into the environment affecting the male reproductive system and male fertility. Sperm motility is one of the initial tests performed to assess sperm function; only motile sperm can achieve fertilization in vivo. The present study aimed to investigate the possible effects of a group of EDs that represent a widespread chemicals in the environment genistein (Gen), is a naturally occurring isoflavone (100 μM), bisphenol A (BPA), that is used in the manufacture of plastics and other products and released largely into the environment (100 μM), nonylphenol (NP) is an important environmental toxicant and potential endocrine disrupting chemical (10 μg/ml), TCDD, that is formed as an unwanted by-product in the manufacture of chlorinated hydrocarbons (2.5 μg/ml), atrazine (Atraz) is a herbicides (500 μM), dibromochloropropane (DBCP) is a pesticide (10 μg/ml), and diazinone (Diaz) is a insecticide (500 μM) on human sperm motility and kinematic characteristics. Human spermatozoa were incubated in Ham's F10 media with/without the tested chemicals or DMSO as positive control for 6 hr at 37℃ in 5% CO2. Then, sperm motility was assessed using computer assisted semen analyzer. Interestingly, all the chemicals tested significantly decreased sperm motility as compared to the control groups. However, only Diaz significantly decreased sperm kinematic characteristics namely, VCL, VSL, STR, VAP, and ALH. We suggest that the environmental chemicals may have an effect on male fertility via decreasing sperm motility.