Do-Yeal Ryu

Increased male fertility using fertility-related biomarkers

Conventional semen analyses are used to evaluate male factor fertility/infertility in humans and other animals. However, their clinical value remains controversial. Therefore, new tools that more accurately assess male fertility based on sperm function and fertilization mechanism are of interest worldwide. While protein markers in spermatozoa that might help differentiate fertile and infertile sperm have been identified, studies are in their infancy, and the markers require validation in field trials. In the present study, to discover more sensitive biomarkers in spermatozoa for predicting male fertility, we assessed protein expression in capacitated spermatozoa. The results demonstrated that cytochrome b-c1 complex subunit 2 (UQCRC2) was abundantly expressed in high-litter size spermatozoa (>3-fold). On the other hand, equatorin, beta-tubulin, cytochrome b-c1 complex subunit 1 (UQCRC1), speriolin, Ras-related protein Rab-2A (RAB2A), spermadhesin AQN-3, and seminal plasma sperm motility inhibitor were abundantly expressed in low-litter size spermatozoa (>3-fold). Moreover, RAB2A and UQCRC1 expression negatively correlated with litter size, while UQCRC2 expression positively correlated with litter size. Finally, the putative biomarkers predicted litter size in field trials. Our study suggests that biomarkers present in spermatozoa after capacitation can help differentiate superior male fertility from below-average fertility with high sensitivity.

Functional and Proteomic Alterations of F1 Capacitated Spermatozoa of Adult Mice Following Gestational Exposure to Bisphenol A

Studies regarding bisphenol A (BPA) exposure and male (in)fertility have conventionally focused on modifications in ejaculated spermatozoa function from exposed individuals. However, mammalian spermatozoa are incapable of fertilization prior to achieving capacitation, the penultimate step in maturation. Therefore, it is necessary to investigate BPA-induced changes in capacitated spermatozoa and assess the consequences on subsequent fertilization. Here, we demonstrate the effect of gestational BPA exposure (50 μg/kg bw/day, 5 mg/kg bw/day, and 50 mg/kg bw/day) on the functions, biochemical properties, and proteomic profiles of F1 capacitated spermatozoa from adult mice. The data showed that high concentrations of BPA inhibited motility, motion kinematics, and capacitation of spermatozoa, perhaps because of increased lipid peroxidation and protein tyrosine nitration, and decreased intracellular ATP levels and protein kinase-A activity in spermatozoa. We also found that BPA compromised the rates of fertilization and early embryonic development. Differentially expressed proteins identified between BPA-exposed and control groups play a critical role in energy metabolism, stress responses, and fertility. Protein function abnormalities were responsible for the development of several diseases according to bioinformatics analysis. On the basis of these results, gestational exposure to BPA may alter capacitated spermatozoa function and the proteomic profile, ultimately affecting their fertility potential.

Peroxiredoxin activity is a major landmark of male fertility

Peroxiredoxins (PRDXs) are important antioxidant enzymes reported to have a role in sperm function and male fertility. However, how PRDXs affects male fertility remain fundamental unanswered questions. We therefore sought to investigate the role of these enzymes in sperm function and fertilisation. In this in vitro trial, mouse spermatozoa were incubated with different concentrations of conoidin A (1, 10, or 100 µM), a specific inhibitor of PRDXs. Our results demonstrated that inhibition of PRDXs by conoidin A significantly decreased the oxidized form of peroxiredoxins (PRDXs-SO3) in spermatozoa. Decreased PRDX activity was associated with a significant reduction in sperm motility parameters, viability, and intracellular ATP, whereas ROS levels, DNA fragmentation, and loss of mitochondrial membrane potential were increased. Simultaneously capacitation and the acrosome reaction were also significantly inhibited perhaps as a consequence of decreased tyrosine phosphorylation and protein kinase-A activity. In addition, fertilisation and early embryonic development were adversely affected following PRDXs inhibition in spermatozoa. Taken together, our data demonstrate that decreased PRDX activity directly affects male fertility due to negative effects on important functions and biochemical properties of spermatozoa, ultimately leading to poor fertilisation and embryonic development.

Elevated aminopeptidase N affects sperm motility and early embryo development

Aminopeptidase N (APN) is a naturally occurring ectopeptidase present in mammalian semen. Previous studies have demonstrated that APN adversely affects male fertility through the alteration of sperm motility. This enzyme constitutes 0.5 to 1% of the seminal plasma proteins, which can be transferred from the prostasomes to sperms by a fusion process. In the present study, we investigated the molecular mechanism of action of APN and its role in regulating sperm functions and male fertility. In this in vitro study, epididymal mouse spermatozoa were incubated in a capacitating media (pH 7) containing 20 ng/mL of recombinant mouse APN for 90 min. Our results demonstrated that the supplementation of recombinant APN in sperm culture medium significantly increased APN activity, and subsequently altered motility, hyperactivated motility, rapid and medium swimming speeds, viability, and the acrosome reaction of mouse spermatozoa. These effects were potentially caused by increased toxicity in the spermatozoa. Further, altered APN activity in sperm culture medium affected early embryonic development. Interestingly, the effect of elevated APN activity in sperm culture medium was independent of protein tyrosine phosphorylation and protein kinase A activity. On the basis of these results, we concluded that APN plays a significant role in the regulation of several sperm functions and early embryonic development. In addition, increased APN activity could potentially lead to several adverse consequences related to male fertility.

Comparison of markers predicting litter size in different pig breeds

To overcome the limitations of conventional analysis of male fertility in animals and humans, proteomic studies have been performed to develop fertility‐related biomarkers for prognosis and diagnosis of male fertility. However, the studies were focused on specific species or breeds. Therefore, a study is required to validate whether fertility‐related markers would apply to other breeds in pigs. In this study, previously developed fertility‐related biomarkers from Landrace were validated to use for prognosis of male fertility in commercially available breeds. Expression level of eight biomarkers in non‐capacitated and capacitated (C) spermatozoa from Yorkshire and Duroc boars was analyzed. And then, to explore the validity of these markers for prognosis of male fertility, i.e. litter size, artificial insemination was performed. Among them, RAB2A (NC) and UQCRC1 (NC) turned out to be highest efficient markers for Yorkshire. RAB2A (C) was most efficient marker for Duroc. Average litter size has increased as much as 1.41 live born after prediction using eight fertility‐related biomarkers in Yorkshire. In addition, average 2.52 litter size was increased after prediction using eight fertility‐related biomarkers in Duroc. Average litter sizes were especially highly increased after prediction of fertility using RAB2A (NC) in Yorkshire (1.57 piglets) and TPI (NC) in Duroc (3.14 piglets), respectively. As a result, all biomarkers were significantly correlated with litter size. However, overall accuracy to predict litter size in three breeds was different in response with each marker. Average litter size after artificial insemination was also significantly affected by marker selection. Therefore, this study suggests that developed fertility‐related markers may be used for prognosis and diagnosis of male fertility irrespective of breed. However, selection of efficient markers for breeds should be considered to obtain more accurate and efficient outcomes.

Applications of capacitation status for litter size enhancement in various pig breeds

Objective Several studies have reported the development of new molecular methods for the prognosis and diagnosis of male fertility based on biomarkers aimed at overcoming the limitations of conventional male fertility analysis tools. However, further studies are needed for the field application of these methods. Therefore, alternative methods based on existing semen analysis methods are required to improve production efficiency in the animal industry. Methods we examined the possibility of improving litter size in various pig breeds using combined Hoechst 33258/chlortetracycline fluorescence (H33258/CTC) staining. The correlation between field fertility and capacitation status by combined H33258/CTC staining in different ejaculates spermatozoa (n = 3) from an individual boar (20 Landrace, 20 Yorkshire, and 20 Duroc) was evaluated as well as overall accuracy. Results The acrosome reacted (AR) pattern after capacitation (%) was positively correlated with the litter size of Landrace, Yorkshire, and Duroc pigs and the overall accuracy was 75%, 75%, and 70% in Landrace, Yorkshire, and Duroc pigs, respectively. The difference (Δ) in AR pattern before and after capacitation was positively correlated with the litter size of Landrace, Yorkshire, and Duroc pigs and the overall accuracy was 80%, 65%, and 55% in Landrace, Yorkshire, and Duroc pigs, respectively. However, the difference (Δ) in capacitated (B) pattern before and after capacitation was negatively correlated with the litter size of Landrace pigs and the overall accuracy was 75%. Moreover, average litter size was significantly altered according to different combined H33258/CTC staining parameters. Conclusion These results show that combined H33258/CTC staining may be used to predict male fertility in various breeds. However, the selection of specific efficiency combined H33258/CTC staining parameters requires further consideration. Taken together, these findings suggest that combined H33258/CTC staining may constitute an alternative method for predicting male fertility until such time as fertility-related biomarkers are further validated.

Effect of endocrine disruptors on the ratio of X and Y chromosome-bearing live spermatozoa

Although equal numbers of X and Y spermatozoa are produced during spermatogenesis, the sex chromosome ratio in ejaculated spermatozoa can be altered by exposure to endocrine-disrupting chemicals (EDCs), which can be reflected by altered sex ratios at birth. Here, we hypothesized EDCs affect sperm functions and viability of X and Y chromosome-bearing human spermatozoa. After exposure to genistein (Gen), bisphenol A (BPA), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), dibromochloropropane (DBCP), and diazinon (Diaz), we evaluated motility, viability, capacitation, and differential viability of X and Y spermatozoa. All EDCs tested altered sperm viability, motility, and capacitation. Interestingly, the Y/X ratio of live spermatozoa was significantly lower in sperm treated with TCDD, DBCP, and Diaz than control spermatozoa. Our results suggest that some of EDCs have larger effects on the viability of Y spermatozoa than X spermatozoa, implicating that a reduction in Y sperm viability may result in a female-biased sex ratio of offspring at birth.

Determination of Highly Sensitive Biological Cell Model Systems to Screen BPA-Related Health Hazards Using Pathway Studio.

Bisphenol-A (BPA) is a ubiquitous endocrine-disrupting chemical. Recently, many issues have arisen surrounding the disease pathogenesis of BPA. Therefore, several studies have been conducted to investigate the proteomic biomarkers of BPA that are associated with disease processes. However, studies on identifying highly sensitive biological cell model systems in determining BPA health risk are lacking. Here, we determined suitable cell model systems and potential biomarkers for predicting BPA-mediated disease using the bioinformatics tool Pathway Studio. We compiled known BPA-mediated diseases in humans, which were categorized into five major types. Subsequently, we investigated the differentially expressed proteins following BPA exposure in several cell types, and analyzed the efficacy of altered proteins to investigate their associations with BPA-mediated diseases. Our results demonstrated that colon cancer cells (SW480), mammary gland, and Sertoli cells were highly sensitive biological model systems, because of the efficacy of predicting the majority of BPA-mediated diseases. We selected glucose-6-phosphate dehydrogenase (G6PD), cytochrome b-c1 complex subunit 1 (UQCRC1), and voltage-dependent anion-selective channel protein 2 (VDAC2) as highly sensitive biomarkers to predict BPA-mediated diseases. Furthermore, we summarized proteomic studies in spermatozoa following BPA exposure, which have recently been considered as another suitable cell type for predicting BPA-mediated diseases.