[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Regulation of lipolysis in muscle is a potential mechanism affecting marbling in beef carcasses and fat accumulation in muscles of humans, which is a known risk factor for type 2 diabetes. Adipose triglyceride lipase-mediated lipolysis is inhibited by G0/G1 switch gene 2 (G0S2) and co-activated by comparative gene identification-58 (CGI-58). In this study, bovine G0S2 and CGI-58 were sequenced, and expressions of these genes were compared among various tissues and in muscles between bulls and steers with different degrees of marbling. The protein coding sequences of bovine G0S2 and CGI-58 revealed breed-specific SNPs, causing two amino acid variations for each protein. Bovine CGI-58 mRNA showed two isoforms from alternative splicing. The G0S2 gene was preferentially expressed in fat and, to a lesser degree, in the liver; whereas, CGI-58 was highly expressed in the muscle and fat (P < 0.05), suggesting their association with lipid metabolism in those tissues. The longissimus dorsi muscle (LM) of steers showed higher FABP4, G0S2 and CGI-58 mRNA expression levels than the LM of bulls, implying the roles of those genes more in marbling of steers than in that of bulls. The G0S2 expression was markedly higher in the intramuscular fat (IMF) (P < 0.001); whereas, the CGI-58 expression was significantly higher in the pure muscle portion of the LM of steers (P < 0.01), suggesting that G0S2 and CGI-58 may regulate IMF and intramyocellular triglycerides, respectively. Taken together, our data suggest that G0S2 and CGI-58 are associated with fat content in bovine species.
[Show abstract][Hide abstract] ABSTRACT: Adipose triglyceride lipase (ATGL), catalyzing the initial step of hydrolysis of triacylglycerol (TAG) in adipocytes, has been known to be inhibited by G0/G1 switch gene 2 (G0S2). In this study, we report the porcine G0S2 cDNA and amino acid sequences as well as the expression level of porcine G0S2. The porcine G0S2 mRNA was abundantly expressed in adipose tissue and liver among various tissues. In adipose tissue, porcine G0S2 expression was 16-fold higher in the fat cell fraction than the stromal vascular fraction. The G0S2 level increased significantly during adipogenesis in vitro and in vivo. These data indicate that G0S2 expression is closely associated with lipid accumulation and adipogenesis. Considering G0S2 as an inhibitor of cell proliferation, the relatively low levels of G0S2 in preadipocytes and adipose tissues of fetal and neonatal pigs compared to adipocytes and adipose tissues of adult pigs may allow the fast cell proliferation rates. Further studies showed that a short-term 24-h fast down-regulated G0S2 expression and increased ATGL expression in adipose tissue; however, a long-term calorie restriction for 8 days had no influence on the level of G0S2 but increased ATGL expression. Therefore, porcine G0S2, which is both a negative regulator of ATGL-mediated lipolysis and cell proliferation in adipose tissue, can be down-regulated in vivo by a short-term 24-h fast followed by increased ATGL-mediated lipolysis.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Proteome Research 07/2012; 11(8):4162-8. DOI:10.1021/pr300248s · 4.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To predict the fertility of frozen-thawed bull spermatozoa, a sperm penetration assay (SPA) using zona-free hamster oocytes was optimized, and the assay results were compared with data from field fertility expressed as the non-return rate (NRR). To increase sperm penetration, the spermatozoa were pre-incubated and coincubated with oocytes in media containing various concentrations of heparin (0 to 50 μg/ml). Coincubation with 10 μg/ml heparin showed the highest sperm penetration (P<0.05); it is considered to be the optimized SPA method. Sperm fertility index values obtained from WSPA were significantly correlated with the historic average NRR of 46 bulls (P<0.01). To determine the normal range for SPA, we established the lower limits of the sperm fertility index and set the cut-off value at 2.55, at which point the NRR was more than 70%, using the receiver operating characteristic curve. The overall accuracy for the 46 bulls was 95.7% (44/46) for both the low and high NRR, with a sensitivity of 95.5% (21/22) and a specificity of 95.8%. This protocol would make it easier to discriminate bulls according to their sperm fertilizing ability.
Journal of Reproduction and Development 04/2012; 58(4):461-6. DOI:10.1262/jrd.11-067H · 1.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mechanism of adipose tissue lipolysis has not been fully elucidated. Greater understanding of this process could allow for increased feed efficiency and reduced fat in poultry. Studies in avian species may provide important insight in developing therapies for human obesity, as lipolytic pathways are highly conserved. Adipose triglyceride lipase (ATGL) cleaves triacylglycols, releasing non-esterified fatty acids (NEFA) into the bloodstream. Glucocorticoids have been shown to elevate circulating NEFA. To determine the regulation of ATGL and regulator proteins comparative gene identification-58 (CGI-58) and G(0)/G(1) switch gene 2 (G0S2) by glucocorticoid, 36 chickens received an injection of dexamethasone (4 mg/kg). Saline was administered to an additional 12 birds to determine any effect of stress during handling. Dexamethasone-injected birds were harvested at 0, 0.5, 1, 2, 4, and 6 h after treatment; saline-treated birds were collected at 4 and 6 h. Abdominal and subcutaneous adipose tissue and blood were collected. Gene and protein expression were analyzed via quantitative real-time PCR and western blot. Compared with the saline group, ATGL expression increased in birds injected with dexamethasone. When dexamethasone response was compared to the untreated group up to 6 h following injection, an increase in ATGL protein was observed as quickly as 0.5 h and increased further from 1 to 6 h. Plasma NEFA and glucose increased gradually from 0 to 6 h, reaching statistical significance at 4 h. These data show that ATGL expression is stimulated by glucocorticoid in a time-dependent manner.
[Show abstract][Hide abstract] ABSTRACT: 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.
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).
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.
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.
Human Reproduction 10/2010; 25(10):2427-33. DOI:10.1093/humrep/deq205 · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To assess whether the differential sperm tail swelling patterns observed following hypo-osmotic shock are useful in discriminating normal sperm from aneuploid sperm.
University research setting.
Fluorescence in situ hybridization (FISH) was combined with hypo-osmotic swelling for a simultaneous assessment of aneuploidy and viability in human spermatozoa.
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.
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.
The sperm cells with tail-tip swelling patterns are related to a low frequency of aneuploidy.
Fertility and sterility 07/2009; 94(3):1012-20. DOI:10.1016/j.fertnstert.2009.04.043 · 4.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
International Journal of Andrology 06/2009; 33(4):604-12. DOI:10.1111/j.1365-2605.2009.00976.x · 3.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 < 0.05) in intracellular GSH concentration before and after. fertilization were observed in 50 M -ME supplements to the IVM medium. Also, increases (P < 0.05) in male pronuclear formation after IVF were observed in same treatment group. In conclusion, supplementing -ME into the IVM medium increased intracellular GSH concentrations and increased fertilization in vitro.