Yoo-Jin Park

The transgenerational impact of benzo(a)pyrene on murine male fertility

BACKGROUND Benzo(a)pyrene (BaP) is an endocrine toxicant that is widely distributed in the environment. The adverse effects of BaP on fertility are well documented, however its effects on fertility in the subsequent generations are not known. We aimed to investigate the transgenerational effects of BaP on male fertility in mice. METHODS Six-week-old male mice (F0) were orally administered BaP (1 or 10 mg/kg body weight) or corn oil, daily for 6 weeks. The male mice were mated with untreated female mice to produce F1 offspring. The F2 and F3 progeny were produced in a similar manner. Testes and spermatozoa were collected from 14-week-old F0, F1, F2 and F3 males in order to assess male fertility parameters, namely testis histology, sperm count, sperm motility and sperm penetration (sperm penetration assay). RESULTS Oral administration of a high dose of BaP induced testicular malformation and decreased numbers of seminiferous tubules with elongated spermatids for three generations studied (i.e. F0 to F2) with significant decreases in F0 and F2. It also significantly decreased sperm motility in F0. BaP significantly decreased sperm count in the group treated with a high dose of BaP in all generations except the F3 generation. The sperm fertility index (SFI) also decreased significantly for two generations. Of the fertility parameters measured, sperm count and SFI were the more sensitive parameters in our study. CONCLUSIONS Exposure to BaP decreases the fertilization potential of exposed males and has an adverse impact on sperm function and fertility in subsequent generations. The BaP effect on fertility can be described as a transgenerational effect for F2 generation.

Fertility-Related Proteomic Profiling Bull Spermatozoa Separated by Percoll

Infertility or subfertility of bovine spermatozoa may lead to disintegration of the breeding system and large economic losses. Recently, proteomics have identified candidates for the sperm fertility biomarkers, but no definite studies have clearly identified the relationship between the proteome and sperm fertility after proteomic study. Therefore, to determine the clinical value of the protein markers identified by proteomic study, we first compared the protein expression profiles of spermatozoa from high and low fertility bulls using 2-dimensional electrophoresis. We then investigated the relationship between protein expression and the fertility of individual bulls as assessed by Western blot analysis. Five proteins, enolase 1 (ENO1), ATP synthase H+ transporting mitochondrial F1 complex beta subunit, apoptosis-stimulating of p53 protein 2, alpha-2-HS-glycoprotein, and phospholipid hydroperoxide glutathione peroxide, were more highly represented in high fertility bulls, whereas three proteins, voltage dependent anion channel 2 (VDAC2), ropporin-1, and ubiquinol-cytochrome-c reductase complex core protein 2 (UQCRC2), were more highly represented in low fertility bulls. Among those proteins, ENO1, VDAC2, and UQCRC2 were significantly correlated with individual fertility. Therefore, these results suggest that concurrent comparisons between protein expression and other fertility assays may represent a good in vitro assay to determine sperm fertility.

Voltage-dependent anion channels are a key factor of male fertility

OBJECTIVE To examine how voltage-dependent anion channels (VDACs) regulate sperm function in capacitation conditions. DESIGN Experimental prospective study. SETTING Academic research laboratory. ANIMAL(S) Male ICR and female B6D2F1/CrljOri mice (8-12 weeks old). INTERVENTION(S) Female mice were superovulated with 5 IU of pregnant mare serum gonadotropin given IP and 5 IU of hCG given IP 48 hours later. Oocytes were applied to assess fertilization and embryo development. MAIN OUTCOME MEASURE(S) Immunofluorescence assay, computer-assisted sperm analysis, hypo-osmotic swelling test, combined Hoechst 33258/chlortetracycline fluorescence assessment of capacitation status, measurement of [Ca(2+)](i) and [pH](i), Western blotting, and IVF. RESULT(S) VDAC2 was localized on the acrosomal region and principal piece, while VDAC3 was localized on the acrosomal region and midpiece. Blocking VDAC with DIDS (500 μM) significantly decreased motility, viability, acrosome reaction, capacitation, tyrosine phosphorylation, fertilization, and embryo development regardless of Ca(2+). However, the most severe decreases were observed in the presence (+) of DIDS and absence (-) of Ca(2+), respectively. A significant decrease in [Ca(2+)](i) concentration was observed in (-) DIDS, while [pH](i) was significantly increased in (-) DIDS regardless of Ca(2+). However, a significantly elevated [pH](i) was observed in (+) Ca(2+). CONCLUSION(S) Abnormal regulation of VDACs negatively affected sperm function. Thus, VDACs may be key regulators of the fertilization ability of spermatozoa.

A comprehensive proteomic approach to identifying capacitation related proteins in boar spermatozoa

BackgroundMammalian spermatozoa must undergo capacitation, before becoming competent for fertilization. Despite its importance, the fundamental molecular mechanisms of capacitation are poorly understood. Therefore, in this study, we applied a proteomic approach for identifying capacitation-related proteins in boar spermatozoa in order to elucidate the events more precisely. 2-DE gels were generated from spermatozoa samples in before- and after-capacitation. To validate the 2-DE results, Western blotting and immunocytochemistry were performed with 2 commercially available antibodies. Additionally, the protein-related signaling pathways among identified proteins were detected using Pathway Studio 9.0.ResultWe identified Ras-related protein Rab-2, Phospholipid hydroperoxide glutathione peroxidase (PHGPx) and Mitochondrial pyruvate dehydrogenase E1 component subunit beta (PDHB) that were enriched before-capacitation, and NADH dehydrogenase 1 beta subcomplex 6, Mitochondrial peroxiredoxin-5, (PRDX5), Apolipoprotein A-I (APOA1), Mitochondrial Succinyl-CoA ligase [ADP-forming] subunit beta (SUCLA2), Acrosin-binding protein, Ropporin-1A, and Spermadhesin AWN that were enriched after-capacitation (>3-fold) by 2-DE and ESI-MS/MS. SUCLA2 and PDHB are involved in the tricarboxylic acid cycle, whereas PHGPx and PRDX5 are involved in glutathione metabolism. SUCLA2, APOA1 and PDHB mediate adipocytokine signaling and insulin action. The differentially expressed proteins following capacitation are putatively related to sperm functions, such as ROS and energy metabolism, motility, hyperactivation, the acrosome reaction, and sperm-egg interaction.ConclusionThe results from this study elucidate the proteins involved in capacitation, which may aid in the design of biomarkers that can be used to predict boar sperm quality.

Discovery of Predictive Biomarkers for Litter Size in Boar Spermatozoa

Conventional semen analysis has been used for prognosis and diagnosis of male fertility. Although this tool is essential for providing initial quantitative information about semen, it remains a subject of debate. Therefore, development of new methods for the prognosis and diagnosis of male fertility should be seriously considered for animal species of economic importance as well as for humans. In the present study, we applied a comprehensive proteomic approach to identify global protein biomarkers in boar spermatozoa in order to increase the precision of male fertility prognoses and diagnoses. We determined that l-amino acid oxidase, mitochondrial malate dehydrogenase 2, NAD (MDH2), cytosolic 5′-nucleotidase 1B, lysozyme-like protein 4, and calmodulin (CALM) were significantly and abundantly expressed in high-litter size spermatozoa. We also found that equatorin, spermadhesin AWN, triosephosphate isomerase (TPI), Ras-related protein Rab-2A (RAB2A), spermadhesin AQN-3, and NADH dehydrogenase [ubiquinone] iron-sulfur protein 2 (NDUFS2) were significantly and abundantly expressed in low-litter size spermatozoa (>3-fold). Moreover, RAB2A, TPI, and NDUFS2 were negatively correlated with litter size, whereas CALM and MDH2 were positively correlated. This study provides novel biomarkers for the prediction of male fertility. To the best of our knowledge, this is the first work that shows significantly increased litter size using male fertility biomarkers in a field trial. Moreover, these protein markers may provide new developmental tools for the selection of superior sires as well as for the prognosis and diagnosis of male fertility.

Vasopressin effectively suppresses male fertility.

Arginine vasopressin (VP) is neurohypophysial hormone has been implicated in stimulating contractile activity of the male reproductive tract in the testis. Higher levels of VP decrease sperm count and motility. However, very little is known about the involvement of VP in controlling mammalian reproductive process. The goal of this study was to confirm that effect of VP receptor (AVPR2) on sperm function in capacitation condition. Deamino [Cys 1, D-ArgS] vasopressin (dDAVP), an AVPR2 agonist that operates only on AVPR2, was used. Also, Mouse spermatozoa were incubated with various concentrations of dDAVP (10−11–10−5 M) and sperm motility, capacitation status, Protein Kinase A activity (PKA), tyrosine phosphorylation, fertilization, and embryo development were assessed using computer-assisted sperm analysis, Combined Hoechst 33258/chlortetracycline fluorescence, Western blotting, and in vitro fertilization, respectively. AVPR2 was placed on the acrosome region and mid-piece in cauda epididymal spermatozoa, but the caput epididymal spermatozoa was mid-piece only. The high dDAVP treatment (10−8 and 10−5 M) significantly decreased sperm motility, intracellular pH and PKA substrates (approximately 55 and 22 kDa) and increased Ca2+ concentration. The highest concentration treatment significantly decreased PKA substrate (approximately 23 kDa) and tyrosine phosphorylation (approximately 30 kDa). VP detrimentally affected capacitation, acrosome reaction, and embryo development. Treatment with the lowest concentration (10−11 M) was not significantly different. Our data have shown that VP stimulates ion transport across sperm membrane through interactions with AVPR2. VP has a detrimental effect in sperm function, fertilization, and embryonic development, suggesting its critical role in the acquisition of fertilizing ability of mouse spermatozoa. These research findings will enable further study to determine molecular mechanism associated with fertility in capacitation and fertilization. It is also an important pivotal precondition to the progress of diagnostic test to identify infertility and to apply male contraception.

Capacitation status of stored boar spermatozoa is related to litter size of sows.

Semen parameters can be considered useful predictors of sperm fertility. The objective of this study was to address the question of whether differences in in vivo fertility after the use of different ejaculates could be predicted using sperm kinematics, capacitation status, and sperm penetration ability under commercial pig production conditions. The percentage of capacitated sperm, as assessed by chlortetracycline (CTC) staining, was positively correlated with litter size (p<0.01). Our data suggest that litter size increases in proportion to the number of capacitated spermatozoa. When all semen parameters (kinematics, sperm capacitation status, and sperm penetration ability) and litter size were included in a multiple linear regression analysis, significant associations were found between the percentage of capacitated sperm (B-type), the sperm fertility index as assessed by a sperm penetration assay (SPA), and litter size. This relationship between capacitated sperm and litter size, however, was more predictive for smaller litter groups than larger ones. We found that the percentage of B-type sperm was significantly correlated with historic average litter size. However, there was no significant correlation between the percentage of B-type sperm and historic farrowing rates. To determine the normal range for B-type sperm, the lower limits were established as 30% for small litters (<8 piglets) and 35% for large litters. The overall accuracy of the assay was 92% and 83% for small and large litters, respectively. These results indicate that capacitation status as measured by CTC staining is a useful predictor of sperm fertility, equivalent to SPA. Moreover, original capacitation status exhibited better predictive ability for small litters than for large ones. Therefore, subfertile boars can be identified primarily by capacitation status.

Xenoestrogenic compounds promote capacitation and an acrosome reaction in porcine sperm

There is growing evidence that endocrine disruptors bind to hormone receptors; since these receptors are present on the sperm membrane, sperm are potentially a useful model for examining estrogenic activities of endocrine disruptors. The objective of the present study was to compare the effects of two xenoestrogenic compounds (genistein and 4-tert-octylphenol) to those of two steroids (estrogen and progesterone) and heparin on in vitro capacitation and the acrosome reaction in a porcine sperm model. Porcine sperm were incubated with various concentrations (0.001-100 μM) of each chemical for 15 or 30 min, and then capacitation and the acrosome reaction were assessed using chlortetracycline. Estrogen and progesterone were considerably more potent than the other chemicals in stimulating capacitation. Estrogen stimulated sperm capacitation at all tested concentrations after 15 min of incubation (P < 0.05), whereas progesterone stimulated sperm capacitation at all tested concentrations after 15 and 30 min (P < 0.05). The effect of genistein on sperm capacitation was comparable with that of estrogen, and it was the most potent in stimulating the acrosome reaction. Genistein stimulated the acrosome reaction at all tested concentrations after 30 min (P < 0.05). However, 4-tert-octylphenol had the least effect on capacitation and the acrosome reaction. In summary, since all chemicals studied effectively altered capacitation and the acrosome reaction, it was concluded that porcine sperm could be a useful model for in vitro screening of potential endocrine disruptors. It was noteworthy that concurrent comparisons to steroids increased the ability to determine estrogenic characteristics of the tested chemicals.

Proteomic revolution to improve tools for evaluating male fertility in animals.

Artificial insemination has been used as a common breeding technique for the rapid dissemination of important genes to improve livestock quality. However, infertility or subfertility in the male leads to the disintegration of the breeding system and large economic losses. Therefore, the development of an accurate diagnostic protocol for male fertility is of critical importance. To this end, many basic laboratory assays have been developed on the basis of semen analysis. Although these assays may provide a preliminary estimate of male fertility, their accuracies are often unacceptably low. Therefore, it is vital to develop new semen analyses that are simple to use and accurate. Proteomic approaches will shed light on understanding sperm physiology and help in developing new diagnostic tools for male fertility. The aim of this study was to review the retrospective semen analyses and prospective proteomic studies of male fertility determination and usefulness of proteomic approaches in diagnosing male fertility potential in animal industry.

Proteomic approaches for profiling negative fertility markers in inferior boar spermatozoa

The ability to predict male fertility is of paramount importance for animal breeding industries and for human reproduction. Conventional semen analysis generally provides information on the quantitative parameters of spermatozoa, but yields no information concerning its functional competence. Proteomics have identified candidates for male fertility biomarkers, but no studies have clearly identified the relationship between the proteome and sperm fertility. Therefore, we performed a proteomic analysis to investigate small and large litter size boar spermatozoa and identify proteins related to male fertility. In this study, 20 proteins showed differential expression levels in small and large litter size groups. Nineteen of these proteins exhibited decreased expression in large litter size samples and increased expression 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. In summary, this is the first study to consider negative fertility biomarkers, and the identified proteins could potentially be used as biomarkers for the detection of inferior male fertility.