ng-Ah You

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.

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.

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.

The sperm penetration assay predicts the litter size in pigs

The prediction of sperm fertility is of paramount importance for breeding animals. Multiple laboratory approaches have been developed for this purpose, but they have yielded equivocal results. The objective of this study was to develop and standardize to a method for predicting fertility in vivo in boars using the in vitro penetration assay. To increase the sensitivity and reduce false-negative results of the assay, each step in the procedure was standardized and quality control was applied. Maximum penetration of hamster zona-free oocytes and immature porcine oocytes was obtained using heparin-treated sperm cells. Hamster zona-free oocytes showed a significantly higher penetration than immature porcine oocytes. To eliminate interassay variability, two frozen bull semen samples were applied. All possible variables related to the female were excluded. The SPA (sperm penetration assay using zona-free oocytes) result showed significant correlation with historic average litter size but had no significant correlation with farrowing rates. To determine the normal range for the SPA, lower limits of the sperm fertility index were established as 1.2 for the small litter sizes (<8 piglets) and 2.5 for the large litter sizes. The overall accuracy was 92 and 96% respectively, for the small and large litter sizes. Our laboratory has standardized the procedure for the SPA, resulting in greatly increased sensitivities for small and large litter sizes. The protocol increases the ability to discriminate between good and poor fertility groups and it was highly effective at ranking 24 boars by litter size into large and small litter groups.

Effect of sodium fluoride on male mouse fertility

Sodium fluoride (NaF), an environmental pollutant, has been tested for its impact on fertility in several species of laboratory animals. A literature demonstrated that NaF adversely affects sperm motility, morphology, capacitation, and the acrosome reaction. However, the molecular mechanisms underlying these alterations have not yet been elucidated. Therefore, present study was designed to evaluate the regulatory pathways involved in the effect of NaF on sperm function and fertilization. In this in vitro study, mouse spermatozoa were incubated with a range of concentrations (2.5, 5, and 10 mm) of NaF for 90 min in media that support in vitro fertilization. Our results showed that NaF was associated with reduced intracellular ATP generation, motility, and motion kinematics. Likewise, short‐term exposure of spermatozoa to NaF significantly reduced the intracellular calcium concentration, protein kinase‐A activity, and tyrosine phosphorylation of sperm proteins, which were associated with a significant decrease in the rate of capacitation and the acrosome reaction. Finally, NaF significantly reduced the fertilization and blastocyst formation during early embryonic development. On the basis of these results, we propose that NaF reduces sperm motility, capacitation, and the acrosome reaction leading to poor fertilization and suppressed embryonic development.

Sodium nitroprusside suppresses male fertility in vitro

Sodium nitroprusside is a nitric oxide donor involved in the regulation of the motility, hyperactivation, capacitation, and acrosome reaction (AR) of spermatozoa. However, the molecular mechanism underlying this regulation has not yet been elucidated. Therefore, this study was designed to evaluate the molecular basis for the effects of sodium nitroprusside on different processes in spermatozoa and its consequences on subsequent oocyte fertilization and embryo development. In this in vitro study, mouse spermatozoa were incubated with various concentrations of sodium nitroprusside (1, 10, and 100 μm) for 90 min. Our results showed that sodium nitroprusside inhibited sperm motility and motion kinematics in a dose‐dependent manner by significantly enhancing intracellular iron and reactive oxygen species (ROS), and decreasing Ca2+, and adenosine triphosphate levels in spermatozoa. Moreover, short‐term exposure of spermatozoa to sodium nitroprusside increased the tyrosine phosphorylation of sperm proteins involved in PKA‐dependent regulation of intracellular calcium levels, which induced a robust AR. Finally, sodium nitroprusside significantly decreased the rates of fertilization and blastocyst formation during embryo development. Based on these results, we propose that sodium nitroprusside increases ROS production and precocious AR may alter overall sperm physiology, leading to poor fertilization and compromised embryonic development.

Nutlin-3a Decreases Male Fertility via UQCRC2

Ubiquinol-cytochrome-c reductase core protein 2 (UQCRC2) is a component of ubiquinol-cytochrome c reductase complex that is known to correlate with male fertility via spermatogenesis. Simultaneously, nutlin-3a is a small molecule antagonist of mouse double minute 2 repressor (MDM2), activate p53 and induce apoptosis responsible for spermatogenesis. To date, however there are no known effects of nutlin-3a on reproduction. Therefore, present study was designed to investigate the effect of nutlin-3a on male fertility via UQCRC2. In this in vitro trial with mice spermatozoa, we utilized CASA, CTC staining, ATP assay, western blotting, and IVF to measure the main study outcome. The short-term exposure of spermatozoa in nutlin-3a decreases sperm motion kinematics, intracellular ATP production, capacitation, the acrosome reaction, UQCRC2, and tyrosine phosphorylation (TYP) of sperm proteins in a dose-dependent manner. Notably, the decreased UQCRC2 and TYP were associated with reduced sperm kinematics, ATP production, and capacitation, which ultimately led to adverse effects on male fertility such as poor fertilization rates and embryo development. Thus, nutlin-3a may be considered as a potential male contraceptive agent due to its ability to decrease fertility secondary to changes in overall sperm physiology and embryonic development. However, the results of this preliminary study have to be confirmed by additional independent trial.