Effect of Aminopeptidase N on functions and fertility of mouse spermatozoa in vitro

Khatun, Amena,Kang, Kyu-Ho,Ryu, Do-Yeal,Rahman, Md Saidur,Kwon, Woo-Sung,Pang, Myung-Geol Elsevier 2018 Theriogenology Vol.118 No.-

<P><B>Abstract</B></P> <P>Aminopeptidase N (APN) is defined as a multifunctional enzyme, which regulate cellular physiology of a wide variety of cells in human. Earlier studies reported that mammalian semen shares this common enzyme as a major protein of seminal plasma that has correlation with male fertility, while the regulatory mechanisms of APN in spermatozoa are still far from being well understood. Present study was designed to investigate the role of APN in biological and chemical functions of spermatozoa using an in vitro antagonistic approach. Results showed that lower APN activity in sperm culture medium significantly increased sperm motility and the percentage of high speed spermatozoa and decreased the percentage of slow speed spermatozoa after a dose dependent inhibitor treatment (10, 100, and 1000 μM leuhistin) on epididymal mouse spermatozoa in a capacitating media for 90 min. Both 100 μM and 1000 μM decreased APN activity, while only 1000 μM decreased cell viability and increased PKA activity significantly compared to control. Nonetheless capacitation status, acrosome reaction status, and lactate dehydrogenase activity were not affected. Intriguingly, the treatment affected embryonic development through decreasing tyrosine phosphorylation of proteins and increasing reactive oxygen species levels. Further <I>in silico</I> analysis revealed associated regulatory proteins, which have critical functional role for male fertility.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Inhibition of APN activity in sperm culture medium significantly increased motility. </LI> <LI> Treatments for APN inhibition have decreased APN activity and increased PKA levels. </LI> <LI> Intriguingly, inhibition of APN activity in sperm culture medium had a detrimental effect on early embryogenesis. </LI> <LI> In silico analysis of this enzyme predicted associated regulatory proteins, which have critical functional role in male fertility. </LI> </UL> </P>

Clinical assessment of the male fertility

( Amena Khatun ),( Md Saidur Rahman ),( Myung-geol Pang ) 대한산부인과학회 2018 Obstetrics & Gynecology Science Vol.61 No.2

The evaluation of infertility in males consists of physical examination and semen analyses. Standardized semen analyses depend on the descriptive analysis of sperm motility, morphology, and concentration, with a threshold level that must be surpassed to be considered a fertile spermatozoon. Nonetheless, these conventional parameters are not satisfactory for clinicians since 25% of infertility cases worldwide remain unexplained. Therefore, newer tests methods have been established to investigate sperm physiology and functions by monitoring characteristics such as motility, capacitation, the acrosome reaction, reactive oxygen species, sperm DNA damage, chromatin structure, zona pellucida binding, and sperm-oocyte fusion. After the introduction of intracytoplasmic sperm injection technique, sperm maturity, morphology, and aneuploidy conditions have gotten more attention for investigating unexplained male infertility. In the present article, recent advancements in research regarding the utilization of male fertility prediction tests and their role and accuracy are reviewed.

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

Rahman, Md Saidur,Kwon, Woo-Sung,Ryu, Do-Yeal,Khatun, Amena,Karmakar, Polash Chandra,Ryu, Buom-Yong,Pang, Myung-Geol American Chemical Society 2018 JOURNAL OF PROTEOME RESEARCH Vol.17 No.1

<P>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.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jprobs/2018/jprobs.2018.17.issue-1/acs.jproteome.7b00668/production/images/medium/pr-2017-00668k_0005.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/pr7b00668'>ACS Electronic Supporting Info</A></P>

The Effect of Conoidin A on Spermatozoa

Do-Yeal Ryu,Ki-Uk Kim,Ye-Ji Kim,Amena Khatun,Md Saidur Rahman,Sung-Jae Yoon,Woo-Sung Kwon,Myung-Geol Pang 한국동물생명공학회(구 한국동물번식학회) 2015 발생공학 국제심포지엄 및 학술대회 Vol.2015 No.10

A novel approach to assessing bisphenol-A hazard using in vitro model system

Md Saidur Rahman,Woo-Sung Kwon,Ye-Ji Kim,Do-Yeal Ryu,Amena Khatun,Sung-Jae Yoon,Myung-Geol Pang 한국발생생물학회 2015 한국발생생물학회 학술발표대회 Vol.2015 No.9

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.

Proteomic profilling of negative fertility-related biomarkers in boar spermatozoa

Woo-Sung Kwon,Md Saidur Rahman,Ye-Ji Kim,Do-Yeal Ryu,Amena Khatun,Sung-Jae Yoon,Myung-Geol Pang 한국발생생물학회 2015 한국발생생물학회 학술발표대회 Vol.2015 No.9

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.

The effects of cryoprotectant on sperm proteome during cryopreservation

Sung-Jae Yoon,Md Saidur Rahman,Ye-Ji Kim,Do-Yeal Ryu,Amena Khatun,Woo-Sung Kwon,Myung-Geol Pang 한국발생생물학회 2015 한국발생생물학회 학술발표대회 Vol.2015 No.9

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.

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

Kwon, Woo-Sung,Shin, Dong-Ha,Ryu, Do-Yeal,Khatun, Amena,Rahman, Md Saidur,Pang, Myung-Geol Asian Australasian Association of Animal Productio 2018 Animal Bioscience Vol.31 No.6

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 (${\Delta}$) 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 (${\Delta}$) 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.

A Comprehensive Proteomic Approach to Identifying Cryo-stress

Sung-Jae Yoon,Ki-Uk Kim,Ye-Ji Kim,Do-Yeal Ryu,Amena Khatun,Md Saidur Rahman,Woo-Sung Kwon,Myung-Geol Pang 한국동물생명공학회(구 한국동물번식학회) 2015 발생공학 국제심포지엄 및 학술대회 Vol.2015 No.10

Correlation between Fertility-Related Biomarkers and Litter Size of Different Breeds of Boar

Woo-Sung Kwon,Won-Ki Pang,Ye-Ji Kim,Do-Yeol Ryu,Amena Khatun,Md Saidur Rahman,Sung-Jae Yoon,Myung-Geol Pang 한국동물생명공학회(구 한국동물번식학회) 2015 발생공학 국제심포지엄 및 학술대회 Vol.2015 No.10