[Objectives] Na⁺/K⁺-ATPase is an integral membrane protein which is present in various cells. This cation antiporter works for the modulation of membrane potential by affecting the cation density near the plasma membrane. Previous studies showed that binding of ouabain (a specific Na⁺/K⁺-ATPase inhibitor) with Na⁺/K⁺-ATPase reduced the motility of rat sperm, and that this antiporter was detected as a band with the molecular mass of 110 kDa on the Western blots of rat sperm proteins. These indicate that Na⁺/K⁺-ATPase has the function in the maintenance of sperm motility. However, roles of Na⁺/K⁺-ATPase in the regulation of bovine sperm motility are not fully understood yet. The objectives of this study are to detect Na⁺/K⁺-ATPase in the bovine sperm and to examine roles of this antiporter in the regulation of maintenance of motility and occurrence of hyperactivation.[Methods] Bovine sperm were collected from mature Japanese Black cattle and used for the detection of Na⁺/K⁺-ATPase by Western blotting and examination of effects of its inhibition by the addition of digoxin to the medium or deficiency of K⁺ in the medium on motility of sperm treated with a cAMP analog to induce full-type hyperactivation.[Results] Bovine sperm Na⁺/K⁺-ATPase was detected as an approximately 105-kDa band. When digoxin (25 nM) was added to the medium to inhibit Na⁺/K⁺-ATPase, the percentages of motile sperm and full-type hyperactivated sperm were significantly higher after 1 or 2 hours of incubation. Moreover, when K⁺ components were removed from the medium to inhibit the Na⁺/K⁺-ATPase, the percentages of motile sperm were significantly higher after 1 or 2 hours of incubation but full-type hyperactivation was rarely induced. These results suggest that the digoxin-sensitive Na⁺/K⁺-ATPase is involved in the regulation of bovine sperm motility during the induction of full-type hyperactivation, and that extracellular K⁺ may be necessary for the occurrence of full-type hyperactivation in bovine sperm.

The present study was conducted to identify the major sexing sperm protein profile of boar and its association with sperm motility. Semen samples were collected from 10 adult (Durc, age: 12–18 month old) and subjected to evaluation of sperm parameters (motility by CASA). As a method for segregation of semen, X-sperm and Y-sperm were separated by attaching ion-specific material to magnetic beads (Son et al., 2015). Sexing sperm was obtained by magnetic bead method, analyzed by two-dimensional SDS-PAGE, and proteins identified by mass spectrometry (electrospray ionization quadrupole time-of-flight). 87 spots were identified in the X-and Y-bearing sperm, equivalent to 55 different proteins. Total protein content and higher ratio of high molecular weight proteins than those collected from X-sperm. Low quality protein and major seminal plasma glycoprotein PSP-1 precursor, as well as spermadhesin (swine, peptide, 133 aa), protein MENT, fibronectin represented 47.3 ± 7% of the total intensity of all spots in X-bearing sperm. Other proteins expressed in the X-bearing sperm included albumin, hypothetical protein, ATP synthase subunit beta, mitochondrial precursor, fibronectin isoform X8, L-amino acid oxidase isoform X1, mitochondrial malate dehydrogenase 2. On the other hand, proteins expressed in the Y-bearing sperms were represented by three major protein fractions with molecular weights around 24–29 kDa (4.25%), 14–21 kDa (16.4%) and below 10 kDa (79.1%). In conclusion, we presently describe the major proteome of boar sexing sperm and significant associations between X- and Y-bearing sperm proteins and semen parameters. Such relationship will serve as the basic for determination of molecular markers of sperm gender in the swine species.

We have demonstrated that the levels of lysosomal Cathepsin B (CTSB) transcript was significantly higher in trophectoderm (TE) than inner cell mass (ICM) of bovine blastocyst, whereas CTSB activity was significantly higher in ICM than TE. In the present study, we investigated the opposite expression pattern of CTSB gene and protein in blastocyst. CTSB protein distribution in IVF derived blastocyst was achieved by immunostaining. Supported by activity, CTSB protein was clearly localized in the cytoplasm and significantly higher in ICM than TE. Therefore, we hypothesized the opposite expression pattern was caused by the different turnover of CTSB by consuming of mRNA in ICM and TE. On day7, the blastocysts were treated by cycloheximide (CHX) for 2 h to block protein synthesis followed by re-cultured in KSOM. After that, total RNA amount was detected by Acridine Orange (AO) staining in whole blastocyst. CHX treatment increased the total RNA amount, especially in ICM. In the next experiment, CTSB was quantified in separated ICM and TE by micro blade dissection after CHX treatment. CTSB abundance was significantly increased in CHX-treated ICM compared to control ICM, whereas no difference in CHX-treated TE than control TE. Importantly, the increasing ratio of CTSB transcript was significantly increased in ICM than TE between CHX-treated and control group. Taken together, these results suggest that low CTSB level in ICM than TE is not caused the down regulation of CTSB gene, but the high turnover of CTSB used for protein synthesis and enzymatic activity for potential role of differentiation.

Synchronization of the estrus of sow using altrenogest treatment has been known to increase ovulation and strong estrus. The success of pregnancy relies on moderate cell-cell signaling environment. FGF-9 and galectin-3, multifunctional proteins, its unique functions are including embryonic development, cell growth, adhesion, migration, invasion, angiogenesis and immune function. The purpose of this study was to investigate the effect if estrus synchronization to altrenogest regumate, PMSG/hCG and artificial insemination (AI) on galectin-3 and FGF-9 genes and protein expression. The morpho-metrical parameters of the endometrium and the number of corpora lutea (CL) were recorded. Crossbred gilts (n = 7) received altrenogest during 18 days [20 mg Regumate (Janssen Animal Health, Beerse, Belgium)], starting 5–7 days after onset of first estrus. Control sows (n = 7) did not receive altrenogest. Endometrium samples, uterine luminal flushing, and ovaries were collected on days 4–5 after AI. The total number of CL was higher (p < 0.05) in the gilt treated with regumate/PMSG/hCG. The content of gelactin-3 and FGF-9 mRNA in pre-embryonic development stages increased on particular days, in control and studied in regumate/PMSG/hCG administered sows. Gelactin-3 and FGF-9 were affected by regumate/PMSG/hCG treatment in the both pre-embryonic development stages (P < 0.001, P < 0.05) and endometrial tissue (P < 0.001, P < 0.01). The regumate/PMSG/hCG treatment resulted in elevated expression of gelactin-3 and FGF-9 (P < 0.005) in ovary tissues in comparison to control sows. Whereas, expression characteristics of gelactin-3 and FGF-9 were investigated by hematoxylin and eosin stained and immunohistochemical staining. The results showed that galectin-3 and FGF-9 were significantly shown in the endometrium and ovary tissues of the regumate/PMSG/hCG. Presented data show that exogenous hormones administration can affect gene and protein expression in the sow reproductive tract.

Mammalian zygotes were found hypersensitive to radiation exposure, which may be ascribed to unusual DNA damage responses. In the present study, we irradiated mouse zygotes with γ-ray at various phases of the cell cycle and examined the developmental effects of irradiation. Zygotes were irradiated in G1 and G2 phases and their cellular responses were assessed by examining the entry into mitosis, micronucleus formation and development to the blastocyst stage. The zygotes irradiated in G1 phase were arrested for longer time than those irradiated in G2 phase. Besides, although pronuclei formed normally in zygotes irradiated in G1 phase, these zygotes had higher tendency to possess micronuclei when they cleaved into the two-cell stage, and lower potential to develop into the blastocyst stage, compared with the zygotes irradiated in G2 phase. These results indicated that the sensitivity of zygotes to irradiation varied between the two gap periods. To investigate the response to irradiation in M phase, zygotes were arrested at M phase by nocodazole treatment. Although the irradiation did not cause any delay in the cleavage into the 2-cell stage, nucleoplasmic bridges, which form due to inappropriate DNA repair and telomere fusions, were frequently observed. Most of these embryos were permanently arrested in the interphase of the two-cell stage and none of them reached the blastocyst stage. Therefore, these results suggested that mitotic checkpoint was not activated by DNA damage in zygotes, the same as what has been observed in somatic cells. However, DNA repair system, which is shut down during somatic mitosis, seemed to be functional but not appropriate during the first mitosis.

The use of estradiol and progesterone value for canine ovulation predication has been previously reported. Here, we tried to compare each of these methods precisely for ovulation detection and seasonal accuracy also. Serum progesterone (P4) and estradiol (E2) level were employed for ovulation determination. The P4 and E2 level were measured by using ECLI (Electro Chemi Luminescence Immunoassay) system. For analysis, blood samples were collected from 30 estrus bitches in each group during pro-estrus and estrus period. We predicted 72 hours (3 days) as ovulation point with E2 peak and 6–15 ng/ml of P4, respectively. Furthermore, the oocytes were collected on the predicted ovulation day. We collected higher percentage of mature oocytes while using E2 (83.3%) as compared to P4 (53.3%). Alternatively, the P4 levels were checked at 72 hours after E2 peak which was found 4–28 ng/ml; much broader range than 6–15 ng/ml. Accuracy of P4 increased from spring (30.76%) to summer (47.92%) and decreased in autumn (37.50%) and winter (29.16%) gradually. Especially, E2 maintained about 50% to 65% whatever the season and temperature. Correlation analyze showed that dynamic of P4 accuracy highly correlated with environment temperate (Rp4=0.862) but E2 could not be affected by the temperature (RE2=0.199). In conclusion, we found that E2 peak method is more precise than that of P4 range, which deciphered the limitation of later method. Moreover, E2 method cannot be affected by measuring system as peak point is always same regardless of measured values in different machines or different environmental temperature. Therefore, we highly recommend the E2 method for ovulation determination in estrus bitches.

Subclinical endometritis (SE) is known as one of the major problems in reproduction of dairy cattle where the risk of the disease is often not noticed due to its silent mode. Bovine endometrium senses and responds to bacteria (their pathogen-associated molecules; PAMs) by the secretion of inflammatory chemokines and cytokines. Our recent results showed that Toll-like receptor 2/4 (TLR2/4) pathway is involved in mediating sperm-bovine endometrial epithelial cells (BEECs) inflammatory process. This study aimed to (1) elucidate the role of peptidoglycan (PGN-SA) and lipopolysaccharide (LPS O55:B5) at extremely low concentrations in BEECs monolayer model, (2) determine how these molecules interact with the sperm-induced response of BEECs, thus possibly interfere their inflammatory response. BEECs monolayers were incubated with each ligand for 24 h independently, prior to their co-culture with/without sperm for 6 h, and cells were harvested for real-time PCR analysis for pro-inflammatory genes TNFA, IL-1B, IL-8, PGES, TLR2, and TLR4. First, the pathologically high doses of PGN (100 ng and 1 mg/ml) and LPS (100 ng and 1 mg/ml) stimulated the mRNA expression of TNFA, IL-1B, IL-8 and PGES, indicating a strong pro-inflammatory response. Sperm alone upregulated TNFA, IL-1B, IL-8 and PGES expressions, while suppressed the TLR2 expression in BEECs. Whereas low doses of (PGN 10 and 100 pg/ml) or (LPS 10 pg/ml) did not induce a significant pro-inflammatory response in BEECs, they suppressed the stimulatory effect of sperm to induce the pro-inflammatory genes in BEECs. The results suggest that low concentrations at 10 pg/ml of PGN (TLR2-ligand) and LPS (TLR4-ligand) can potentially compete with sperm for activating its specific receptors of BEECs, thereby disrupting the intact immune response to sperm. This may underline the possible mechanisms for SE partly mediated by TLR2/4 system in the cow.