Shin-Ae Oh

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.

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.

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.

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.

Numerical chromosome abnormalities are associated with sperm tail swelling patterns

OBJECTIVE To assess whether the differential sperm tail swelling patterns observed following hypo-osmotic shock are useful in discriminating normal sperm from aneuploid sperm. DESIGN Prospective study. SETTING University research setting. INTERVENTION(S) Fluorescence in situ hybridization (FISH) was combined with hypo-osmotic swelling for a simultaneous assessment of aneuploidy and viability in human spermatozoa. MAIN OUTCOME MEASURE(S) FISH for chromosomes 1, 13, 18, 21, X, and Y after hypo-osmotic stress was used to investigate the distribution of sperm aneuploidy related to sperm-tail swelling patterns. A total of 16,473 sperm cells were scored from three normal fertile donors and six oligoasthenoteratozoospermic (OAT) patients. RESULT(S) There was a 17.2-fold decrease in the frequency of total aneuploidy in the sperm with a tail-tip swelling pattern compared with the initial nonselected sperm in the OAT patients. Strikingly, when the sperm with tail-tip swelling patterns were screened from the patients, the frequency of total aneuploidy was actually lower by a factor of four than in the nonselected sperm from fertile donors. CONCLUSION(S) The sperm cells with tail-tip swelling patterns are related to a low frequency of aneuploidy.

Effect of β-Mercaptoethanol Supplement during In Vitro Maturation on IVM, IVF and Glutathione Level in Porcine Oocytes

Experiments were conducted to determine the effects of beta-mercaptoethanol (-ME) supplements to the in vitro maturation (IVM) medium on in vitro fertilization (IVF) and intracellular glutathione (GSH) concentration. Porcine cumulus-intact oocytes were matured in TCM-I99 medium containing porcine follicular fluid, sodium pyruvate, D-glucose, FBS, hormonal supplements, and -ME (0, 25, 50 and 100 M) for 36 to 46h. After culture, cumulus-free matured oocytes were co-incubated with epididymal spermatozoa for 18h. There were no significant differences in the maturation rate among treatment groups. However, increases (P M -ME supplements to the IVM medium. Also, increases (P -ME into the IVM medium increased intracellular GSH concentrations and increased fertilization in vitro.