• Journal of Microbiology and Biotechnology
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• v.25 no.11
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• pp.1944-1953
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• 2015

A novel alkaline protease from Streptomyces sp. M30, SapHM, was purified by ammonium sulfate precipitation, hydrophobic interaction chromatography, and DEAE-Sepharose chromatography, with a yield of 15.5% and a specific activity of 29,070 U/mg. Tryptic fragments of the purified SapHM were obtained by electrospray ionization quadrupole time-of-flight mass spectrometry. Nucleotide sequence analysis revealed that the gene sapHM contained 1,179 bp, corresponding to 392 amino acids with conserved Asp156, His187, and Ser339 residues of alkaline protease. The first 24 amino acid residues were predicted to be a signal peptide, and the molecular mass of the mature peptide was 37.1 kDa based on amino acid sequences and mass spectrometry. Pure SapHM was optimally active at 80℃ in 50 mM glycine-NaOH buffer (pH 9.0), and was broadly stable at 0-50℃ and pH 4.0-9.0. The protease relative activity was increased in the presence of Ni²⁺, Mn²⁺, and Cu²⁺ to 112%, 113%, and 147% of control, respectively. Pure SapHM was also activated by dimethylformamide, dimethyl sulfoxide, Tween 80, and urea. The activity of the purified enzyme was completely inhibited by phenylmethylsulfonyl fluoride, indicating that it is a serine-type protease. The K_m and V_max values were estimated to be 35.7 mg/ml, and 5 × 10⁴ U/mg for casein. Substrate specificity analysis showed that SapH was active on casein, bovine serum albumin, and bovine serum fibrin.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.12
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• pp.1820-1826
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• 2007

In this study, we conducted various experiments in order to develop enhanced cultural conditions for in vitro-produced porcine embryos. All embryos were produced by in vitro maturation (IVM) and fertilization (IVF) of immature oocytes from abattoir-derived ovaries. In experiment 1, we cultured IVF embryos in 4 different groups, namely, 0% bovine serum albumin (BSA), 3% BSA, 0.05% Polyvinyl alcohol (PVA), and 0.5% Polyvinylpyrrolidone (PVP) added to the basal fluid cultural medium, Porcine zygote medium 3 (PZM-3). The rates of embryo development were higher in the group where the PZM-3 media had been supplemented with 3% BSA than the other groups. While not statistically significant, the percent of blastocysts and hatched blastocytes were 6.9% and 25.0% in the 3% BSA group vs. 1.2-6.4% and 0-16.7% in the other groups, respectively. In experiment 2, we added 10% fetal bovine serum (FBS) to PZM-3 on day 0 of culture and observed the development rate of blastocysts per day of culture from days 0 to 5. The development rate of blastocysts was higher at 15.6% on day 4 than on any other day, and was significantly higher than on day 0 or day 1 (p<0.05). The development rate of hatched blastocysts was 26.7% on day 4, and was higher than on any other day. In experiment 3, we cultured IVF embryos with different fluid culture media, grouped as 1) PZM-3+0.3% BSA (day0-day7); 2) PZM-3+0.3% BSA${\rightarrow}$day-4) PZM-3+10% FBS; 3) PZM-3+0.3% BSA${\rightarrow}$PZM-3+0.3% BSA+(day-4) FBS 10%; and 4) PZM-3+0.3% BSA+10% FBS (day0-day7). The development rates of blastocysts and hatched blastocysts were 21.5% and 53.1% in group 3, respectively, which was significantly higher than group 4 with respect to blastocyst development (5.2%, p<0.05) but not hatched blastocysts (14.3%). The total cell number (TCN) of blastocysts in group 3 was higher at $37.8{\pm}16.1$ than the other groups at $16.8{\pm}4.4$ - $30.1{\pm}10.9$; however, this was not significantly different. The results of this study showed that PZM-3 containing 0.3% BSA and supplemented with FBS during the later stage of culture on day 4 resulted in better TCNs and an increased rate of hatched blastocysts.

• Korean Journal of Animal Reproduction
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• v.27 no.2
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• pp.169-177
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• 2003

This study was conducted to determine effects of polyvinyl alcohol (PVA), fetal bovine serum (FBS) bovine serum albumin (BSA) and epidermal growth factor (EGF) on blastocoel formation, total cell number, apoptosis and apoptosis-related gene expression of porcine diploid parthenotes developing in vitro. The addition of 0.4% BSA to the culture medium enhanced the development of 2-cell stage parthenotes to the blastocysts stage (P<0.01). FBS reduced cell numbers of blastocysts (P<0.01) and increased percentage of apoptosis in the blastocysts (P<0.001). However, while BSA increased cell numbers, it did so only when EGF was present. Either agent on its own had no effect. Similarly, apoptosis in the blastocysts was not influenced by either agent on its own but was reduced when both BSA and EGF were present. Furthermore, semi-quantitative reverse-transcriptase polymerase chain reaction revealed that EGF enhanced the mRNA expression of Bcl-xL in the presence of 0.4% BSA but BSA and EGF alone had no effect, and EGF and/or BSA did not influence Bak gene expression in the blastocyst stage parthenotes. However FBS reduced Bcl-xL mRNA expression (P <0.05) and enhanced Bak expression. This result suggests that apoptosis related genes expression is significantly affected by supplements, which may result in alteration of apoptosis and embryo viability of porcine embryos developing in vitro.

• Journal of Embryo Transfer
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• v.29 no.3
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• pp.201-206
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• 2014

The objectives of the study were to determine an effective culture dish, culture duration and protein supplementation in medium for in vitro maturation (IVM) of oocytes of native zebu cows in Bangladesh. The ovaries of cows were collected from local slaughterhouse followed by aspiration of follicular fluid. The cumulus-oocyte-complexes (COCs) with more than 3 compact cumulus cell layers were cultured in tissue culture medium (TCM) 199 for maturation. The maturation of oocytes was determined by observing polar body under microscope. To determine an effective culture dish, 130 COCs derived from 48 ovaries in a well of 4-well dish and 102 COCs derived from 36 ovaries in drops covered with mineral oil within 35 mm petri dish were cultured for 24 hours. The rate of maturation of oocytes did not vary between 4-well dish ($51.3{\pm}15.0%$) and drops in petri dish ($52.4{\pm}11.6%$). To determine the effective culture duration, 185 COCs derived from 62 ovaries were cultured in drops for 18, 21, 24 and 27 hours. The rate of maturation of occytes ranged from $51.9{\pm}9.4%$ (18 hours) to $59.0{\pm}17.1%$ (27 hours) and the difference in maturation rate among different culture durations was not significant (P>0.05). To determine an effective protein supplementation, 63 oocytes from 19 ovaries were cultured separately in TCM 199 supplemented with either fetal bovine serum (FBS) or bovine serum albumin (BSA). The rate of maturation was significantly (P<0.01) higher in medium supplemented with FBS ($55.63{\pm}16.19%$) than that of BSA ($14.82{\pm}9.36%$). In conclusion, COCs of native zebu cows can be cultured for IVM either in 4-well culture dish or droplets in petri dish for 18 to 27 hours in medium supplemented with FBS.

• Journal of Veterinary Science
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• v.24 no.1
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• pp.4.1-4.16
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• 2023

Background: In vitro culture of preantral follicles is a promising technology for fertility preservation. Objectives: This study aims to investigate an optimized three-dimensional (3D) fetal bovine serum (FBS)-free preantral follicle culture system having a simple and easy operation. Methods: The isolated follicles from mouse ovaries were randomly divided in an ultra-low attachment 96-well plates supplement with FBS or bovine serum albumin (BSA) culture or encapsulated with an alginate supplement with FBS or BSA culture. Meanwhile, estradiol (E₂) concentration was assessed through enzyme-linked immunosorbent assay of culture supernatants. The diameter of follicular growth was measured, and the lumen of the follicle was photographed. Spindle microtubules of oocytes were detected via immunofluorescence. The ability of oocytes to fertilize was assessed using in vitro fertilization. Results: The diameters were larger for the growing secondary follicles cultured in ultra-low attachment 96-well plates than in the alginate gel on days 6, 8, and 10 (p < 0.05). Meanwhile, the E2 concentration in the BSA-supplemented medium was significantly higher in the alginate gel than in the other three groups on days 6 and 8 (p < 0.05), and the oocytes in the FBS-free system could complete meiosis and fertilization in vitro. Conclusions: The present study furnishes insights into the mature oocytes obtained from the 3D culture of the preantral follicle by using ultra-low attachment 96-well plate with an FBS-free system in vitro and supports the clinical practices to achieve competent, mature oocytes for in vitro fertilization.

• Korean Journal of Food Science and Technology
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• v.30 no.4
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• pp.988-991
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• 1998

Plasma proteins were obtained from bloods of slaughtered bovine and porcine and analyzed by Fast Protein Liquid Chromatography (FPLC). Serum albumin content decreased in the following order: Porcine Plasma Protein (PPP)> Bovine Plasma Protein (BPP)> Whey Protein Concentrate (WPC). Protein contents of BPP, PPP, and WPC determined by Kjeldahl method were 85.79%, 82.30%, and 84.38%, respectively. Compared to WPC, plasma proteins had higher emulsifying activity index (EAI) below 2% protein concentration and slightly lower EAI above 4% protein concentration. Plasma proteins had higher EAI in the acidic pH range and more dependence on NaCl than WPC. Also, EAI of plasma proteins with NaCl was higher in the acidic range than that of WPC. These results indicated that plasma protein can be utilized as a raw material for emulsifier.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.1
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• pp.95-102
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• 1996

This study was performed to investigate the effect of human follicular fluid (HFF) on development of mouse embryos, for evaluating the suitability of HFF as a substitutive material of human fetal cord serum in ART program. The various concentrations of HFF were added into the culture medium and the effects of HFF concentrations were examined to identify the optimal concentration of HFF for embryo development. The potency of HFF in improving embryo development was compared to that of other protein supplement. Collected HFFs were classified with the maturity of the containing oocytes; mature, immature, atretic, and then the effects of the classified HFFs on embryo development were examined. Also, HFF was separated into the low (<30,000 Da) and high (>30,000 Da) molecular weight fractions and the effects of the fractions on embryo development were investigated. The highest development rate was found in culture medium supplemented with 20% HFF, bnt this rate was reversely reduced at the concentrations of HFF higher than 20%. The development rates to the blastocyst, hatching blastocyst, attachment and outgrowth cultured in mature HFF was significantly higher than those in immature and atretic HFF, and mean cell number in blastocyst was higher in mature HFF than in immature and atretic HFF. The development rates of mouse embryos according to protein sources were significantly higher in HFF than in fetal cord serum (FCS), maternal serum (MS) and bovine serum albumin (BSA), and mean cell number in blastocyst cultured in HFF was higher than that in FCS, MS and BSA. The development rates of embryo and mean cell number in blastocyst cultured in high molecular weight fraction of HFF were higher than those in low molecular weight fraction, but the results of high molecular weight fraction were lower than those of whole HFF. Therefore, these results indicated that human mature follicular fluid was useful for improving the development of mouse embryos, which suggests a possibility that HFF also may be used efficiently for improving the culture condition in human ART program as a protein supplement.

• Clinical and Experimental Reproductive Medicine
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• v.21 no.2
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• pp.183-190
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• 1994

Purpose of the present study was to find the optimal culture conditions for the maturation and fertilization of immature oocytes by the use human body fluids and gonadotropins (Gn) in the mouse model. Cumulus-enclosed mouse immature oocytes were incubated in the medium containing various human body fluids with or without Gn in vitro, and examined to confirm nuclear maturation (NM) and fertilization. Female ICR mice were stimulated with 7.5 IU pregnant mares' serum gonadotropin (PMSG). Cumulus-enclosed immature oocytes were isolated at 48-52 hr post PMSG injection and cultured in TCM 199 supplemented with various concentrations (20, 50, and 70%) of human body fluids such as fetal cord serum (hCS), follicular fluid (hFF), peritoneal fluid (hPF) and amniotic fluid (hAF) in the presence or absence of 10 IU/ml PMSG and 10 IU/ml human chorionic gonadotropin (hCG) for 18 hr. Fetal calf serum (FCS) was used as a control for the supplements. Matured oocytes were fertilized with sperm collected from the epididymis of male mice. Fertilization was conducted in T6 medium containing 15 mgl ml bovine serum albumin, and confirmed at 6 hr post-insemination. Evaluation of nucler maturation and fertilization was carried out by rapid staining using fuchin. There was no significant difference between the effects of human body fluids and FCS supplements on nuclear maturation of cumulus enclosed mouse immature oocytes. When maturation medium was supplemented with 20% hPF or 20% hAF, fertilization rates were significantly (P<0.01) lower than that of 20% FCS, hCS and hFF groups. However, higher concentrations of body fluids during IVM were not more beneficial on fertilizability of oocytes. The addition of Gn significantly increased the fertilization rates in hPF and hAF groups (hPF without Gn; 51.5%, compared with 85.1% for addition of Gn, and hAF without Gn; 30.1% compared with 85.8% for addition of Gn) at 20% concentration. These results suggest that human body fluids at 20% concentration and gonadotropins can be used as supplements for the maturation of mouse immature oocytes in vitro. When gonadotropins supplemented with the human body fluids in the maturation medium, fertilizability of mouse immature oocytes was increased in hPF and hAF groups. These results can be applied to maturation of human immature oocytes in vitro.

• Journal of fish pathology
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• v.22 no.2
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• pp.167-172
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• 2009

Antibody-detection enzyme-linked immunosorbent assay (ELISA) was performed to determine whether the absorbance value of ELISA is influenced by the different serum storage conditions of olive flounder Paralichthys olivaceus. Flounder antiserum to bovine serum albumin was stored at -80, -20, 4 and 20${^{\circ}C}$ for 1, 30, 69 and 124 days, respectively. In addition, the flounder antisera were frozen at -80 and -20${^{\circ}C}$, respectively and then repeated 1, 5 and 10 thaw-freeze cycles. No significant difference was shown in ELISA optical density (O.D.) values of sera, which were stored at the above mentioned storage conditions during 124 days. ELISA O.D. values of unfrozen serum samples, which were previously stored at 4${^{\circ}C}$, were almost similar to those of sera undergoing 1, 5, and 10 freeze-thaw cycles after stored at -80 or -20${^{\circ}C}$. In conclusion, the ELISA O.D. values of flounder sera were not affected by various storage conditions: different temperatures (-80, -20, 4 and 20${^{\circ}C}$), durations of storage (1, 30, 69 and 124 days), and repeated thaw-freeze cycles (1, 5, and 10 times).

• Animal cells and systems
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• v.5 no.1
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• pp.51-57
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• 2001

We have previously shown that oocyte maturation is induced by an immobilized progesterone, progesterone-3-carboxymethyloxime - bovine serum albumin conjugate (P-BSA) in Rana dybowskii. In this study, we confirmed the maturation inducing activity of P-BSA on Xenopus oocyte and examined the binding character of the immobilized progesterone on the surface of Xenopus oocytes after removal of the vitelline layer. P-BSA induced maturation of Xenopus oocytes but E-BSA failed to do so as observed in Rana. Binding of the immobilized progesterone, fluorescein isothiocyanate-labeled progesterone-3-0-carboxymethyloxime-BSA (P-BSA-FITC) on the devitellined oocytes surface was examined by fluorescence confocal microscopy. The binding affinity of P-BSA-FITC to the devitellined oocyte was higher than that of estrogen-BSA-FITC (E-BSA-FITC) or testosterone-BSA-FITC (T-BSA-FITC). The binding disappeared in the presence of excess free progesterone but not in the presence of free estrogen. Maximum binding occurred after two-hours of incubation with P-BSA-FITC at pH 7.5. Stronger binding occurred in oocytes at stage Vl than stage IV, and in vitro treatment of hCG enhanced the binding. Taken together, these results suggest that a specific receptor for progesterone exists on the plasma membrane of Xenopus oocytes and that progesterone acts initially on this putative receptors and triggers generation of membrane-mediated second messengers during the early stage of oocyte maturation In amphibians.