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.

Cryopreservation allows for the advances of the reproductive technique and livestock industry. However, cryopreservation inevitably causes various types of stress, such as cold shock, osmotic stress, and ice crystal formation, thereby reducing fertility. Although cryoprotectant agent (CPA) is added to protect spermatozoa from freezing damage during cryopreservation, it has intrinsic toxicity that can affect components of the sperm membrane. Moreover, the addition of CPA induces osmotic stress and excessive reactive oxygen species (ROS) generation, resulting in disruption of mitochondrial membrane potential, alteration of membrane permeability, and damage of sperm surface proteins. To identify the effects of CPA to spermatozoa, we analyzed the sperm movement, capacitation status, and viability using computer-assisted sperm analysis and Hoechst 33258/chlortetracycline fluorescence staining. Moreover, we performed two-dimensional electrophoresis to find protein markers related CPA addition in cryo processes. CPA addition reduced sperm motility (%), viability (%), and non-capacitated spermatozoa, whereas acrosome-reacted spermatozoa increased significantly (p<0.05). Following addition of CPA, a total of ten proteins were altered their expression (eight increased, two decreased) (>3 fold, p<0.05). Among these, four differentially expressed proteins were related to several canonical pathways, such as the ephrinR-actin, ROS metabolism, actin cytoskeleton assembly, actin cytoskeleton regulation, and respiratory chain and oxidative phosphorylation pathway (p<0.05). The present study suggests that CPA significantly alters the functions and proteome content of spermatozoa. Additionally, we anticipated that the differentially expressed proteins might consider as biomarker of CPA-induced stress.

Prognosis and diagnosis of male fertility is a most important for animal breeding system and human reproduction. Conventional semen analysis generally provides information on the quantitative parameters of spermatozoa, but yields no information concerning its functional competence. Thus, new methods for diagnosis and prognosis of male fertility will need to be developed to ensure more accurate assessments. Proteomics have used to find candidate biomarkers for male fertility, but the relationship between the proteome and fertility was not fully understood. Therefore, we performed a comprehensive proteomic approach to investigate small and large litter size boar spermatozoa and identify proteins related to negative male fertility. In present study, 20 proteins showed differential expression levels in small and large litter size groups. Nineteen of these proteins were abundantly expressed in the small litter group. Interestingly, only one protein was highly expressed in the large litter size spermatozoa. We then identified signaling pathways associated with the differentially expressed protein markers. Glutathione S-transferase Mu3 and glutathione peroxidase 4 were related to the glutathione metabolic pathway and arginine vasopressin receptor 2 was linked to vasopressin R2/STAT. Taken together, our results suggest that identified negative fertility-related biomarkers may be used as negative biomarkers for the detection of inferior male fertility such as sub-fertility or infertility.

Although the toxicological impacts of the xenoestrogen bisphenol-A (BPA) have been studied extensively, but its mechanism of action is poorly understood. Eventually, no standard method exists for evaluating the possible health hazard of BPA. Considering mice spermatozoa as a potential in vitro model, here we demonstrated the effects of BPA exposure (0.0001, 0.01, 1, and 100 μM for 6 h) on spermatozoa and the related mechanisms of action. Our results demonstrated that high concentrations of BPA negatively affect sperm motility, viability, intracellular ATP, and mitochondrial functions by activating the mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and protein kinase-A pathways. The same doses were also employed to identify the differential expressed proteins of exposure and screen their functional affiliation to diseases using sperm proteomics and informatics, respectively. Our results demonstrated that a high concentration of BPA (100 μM) induced differential expression (> 2-fold) of 24 proteins in spermatozoa (16 down- and 9 up-regulated), that are putatively involved in the pathogenesis of several diseases. To the best of our knowledge, this is the first study to demonstrate the mechanisms of BPA action in spermatozoa and to identify the possible biomarkers of exposure. Moreover, we anticipated that current strategy might apply for the hazard assessment of other toxicological agents.

As an endocrine disruptor, bisphenol-A (BPA) causes several functional and behavioral abnormalities related to reproduction. The current study was design to evaluate the effect of perinatal exposure of female mice to BPA on sperm function of adult F(1) offspring. Pregnant female mice F(0) were gavaged with three different concentration of BPA, such as 50 μg/kg/day (tolerable daily intake value by the European Food Safety Authority), 5 mg/kg/day (no-observed-adverse-effect level; NOAEL), and 50 mg/kg/day (lowest-observed-adverse-effect level; LOAEL) and corn oil (7 mg/kg/day; vehicle control). The functional parameters of F(1) spermatozoa were studied both before and after capacitation, whereas the fertility assessment was evaluated by in vitro and in vivo assay using unexposed females. Our results showed that spermatozoa hyperactivated motility, capacitation, intracellular ATP, Ca2+, and ROS levels after capacitation were significantly affected using NOAEL and LOAEL concentration of BPA. However, the sperm motility was only affected by LOAEL dose after capacitation. All of the tested parameters were potentially unaffected by BPA before capacitation, except intracellular ATP that decreased by all concentrations. Although both NOAEL and LOAEL concentration were effectively reduced the rate of fertilization and embryonic development in vitro, however the average litter size was only affected by LOAEL dose. Our finding suggested that perinatal exposure of 50 μg/kg/day did not produce significant effects; however both NOAEL and LOAEL affects overall sperm function after capacitation, leading to impairments in the fertility of F(1) male offspring.

The present study aims to investigate the effect of BPA on sperm functions, fertilization and to evaluate their association with the activity of fertility related proteins in spermatozoa. We used a comprehensive in vitro test system to evaluate the effect of various concentrations of BPA (0.0001, 0.01, 1, and 100 μM) on mouse spermatozoa following 6 h of incubation. Our results showed that high concentration of BPA inhibited sperm motility and motion kinematics by significantly decreasing ATP levels in spermatozoa. Simultaneously, exposure of spermatozoa to high concentrations of BPA increased the tyrosine phosphorylation of sperm proteins involved in PKA-dependent regulation and induced a robust AR, ultimately results in poor fertilization and compromised embryonic development. Finally, BPA effects on selected group of fertility related proteins in spermatozoa, such as it degraded the β-actin, whereas the levels of peroxiredoxin-5, glutathione peroxidase, glyceraldehyde-3-phosphate dehydrogenase, and succinate dehydrogenase were increased. Based on these results, we propose that high concentration of BPA may alter overall sperm functions, fertilization and embryonic development, in association with degradation and/or phosphorylation of fertility related proteins in spermatozoa.