• Journal of Animal Reproduction and Biotechnology
• /
• v.34 no.1
• /
• pp.10-19
• /
• 2019

Morphology of cumulus-oocyte-complexes (COCs) at germinal vesicle (GV) stage as one of the evaluation criteria for oocyte maturation quality after in vitro maturation (IVM) plays important roles on the meiotic maturation, fertilization and early embryonic development in pigs. When cumulus cells of COCs are insufficient, which is induced the low oocyte maturation rate by the increasing of reactive oxygen species (ROS) in porcine oocyte during IVM. The ROS are known to generate including superoxide and hydrogen peroxide from electron transport system of mitochondria during oocyte maturation in pigs. To regulate the ROS production, the cumulus cells is secreted the various antioxidant enzymes during IVM of porcine oocyte. Our previous study showed that Mito-TEMPO, superoxide specific scavenger, improves the embryonic developmental competence and blastocyst formation rate by regulating of mitochondria functions in pigs. However, the effects of Mito-TEMPO as a superoxide scavenger to help the anti-oxidant functions from cumulus cells of COCs on meiotic maturation during porcine oocyte IVM has not been reported. Here, we categorized experimental groups into two groups (Grade 1: G1; high cumulus cells and Grade 2: G2; low cumulus cells) by using hemocytometer. The meiotic maturation rate from G2 was significantly (p < 0.05) decreased (G1: $79.9{\pm}3.8%$ vs G2: $57.5{\pm}4.6%$) compared to G1. To investigate the production of mitochondria derived superoxide, we used the mitochondrial superoxide dye, Mito-SOX. Red fluorescence of Mito-SOX detected superoxide was significantly (p < 0.05) increased in COCs of G2 compared with G1. And, we examined expression levels of genes associated with mitochondrial antioxidant such as SOD1, SOD2 and PRDX3 using a RT-PCR in porcine COCs at 44 h of IVM. The mRNA levels of three antioxidant enzymes expression in COCs from G2 were significantly (p < 0.05) lower than COCs of G1. In addition, we investigated the anti-oxidative effects of Mito-TEMPO on meiotic maturation of porcine oocyte from G1 and G2. Meiotic maturation and mRNA levels of antioxidant enzymes were significantly (p < 0.05) recovered in G2 by Mito-TEMPO ($0.1{\mu}M$, MT) treatment (G2: $68.4{\pm}3.2%$ vs G2 + MT: $73.9{\pm}1.4%$). Therefore, our results suggest that reduction of mitochondria derived superoxide by Mito-TEMPO may improves the meiotic maturation in IVM of porcine oocyte.

• Nutrition Research and Practice
• /
• v.16 no.3
• /
• pp.314-329
• /
• 2022

BACKGROUND/OBJECTIVES: Oocyte lipid droplets play a crucial role in meiosis and embryo development. Biotin is associated with fatty acid synthesis and is the coenzyme for acetyl-CoA carboxylase (ACC). The effects of a biotin deficiency on the oocyte lipid metabolism remain unknown. This study examined the effects of a biotin deficiency and its replenishment on murine 1) oocyte lipid droplet levels, 2) ovary lipid metabolism, and 3) oocyte meiosis. MATERIALS/METHODS: Mice were divided into 3 groups: control, biotin deficient (BD), and recovery groups. The control and BD groups were fed a control diet or BD diet (0.004 or 0 g biotin/kg), respectively. The recovery group mice were fed a BD diet until day 21, and were then fed the control diet from days 22 to 64. This study then quantified the oocyte lipid droplet levels, assessed the oocyte mitochondrial function, and examined the ability of oocytes to undergo meiosis. Ovarian phosphorylated ACC (p-ACC), lipogenesis, β-oxidation, and ATP production-related genes were evaluated. RESULTS: The BD group showed a decrease in lipid droplets and mitochondrial membrane potential and increased p-ACC levels. In the recovery group, the hepatic biotin concentration, ovarian p-ACC levels, and mitochondrial membrane potential were restored to the control group levels. On the other hand, the quantity of lipid droplets in the recovery group was not restored to the control levels. Furthermore, the percentage of oocytes with meiotic abnormalities was higher in the recovery group than in the control group. CONCLUSIONS: A biotin deficiency reduced the oocyte lipid droplet levels by downregulating lipogenesis. The decreased lipid droplets and increased oocyte meiosis failure were not fully restored, even though the biotin nutrition status and gene expression of lipid metabolism was resumed. These results suggest that a biotin deficiency remains robust and can be long-lasting. Biotin might play a crucial role in maintaining the oocyte quality.

• 대한생식의학회:학술대회논문집
• /
• 1996.06a
• /
• pp.23-33
• /
• 1996

• Journal of Embryo Transfer
• /
• v.15 no.3
• /
• pp.287-293
• /
• 2000

This study was undertaken to access the effects of collection method, room temperature at oocyte recovery and culture media on the oocyte quality, fertilization and cleavage rates of in vitro matured and fertilized oocytes of Korean native goats. Ovaries obtained from a slaughterhouse were transported to the laboratory and were divided into 2 groups. One group of ovaries was maintained at 30 to 35$^{\circ}C$ of the room temperature and another group was remained at 20 to $25^{\circ}C$ during oocyte recovery. The oocytes were recovered by follicle aspiration, slicing and aspiration+slicing methods from 3 groups of follicles according to size; <2 mm, 2 to 6 mm and >6 mm. The matured oocytes were inseminated with buck epididymal spermatozoa at a concentration of 3~3.5$\times$10$^{6}$ m1 and fertilization was identified when 2 pronuclei were present in the cytoplasm. Although the recovery rate per ovary obtained by the combination of follicle aspiration + slicing(19.6$\pm$2.2) method was higher than aspiration(11.7$\pm$1.1) and slicing(14.8$\pm$1.8) collection, optimal recovery according to oocyte grades resulted form ovarian slicing compared to aspiration or combined methods(P<0.05). However, no significant differences were found in the mean number(2.5$\pm$1.8; 3.3$\pm$3.3; 2.9$\pm$2.4) and the proportion of favorable oocytes(Grades I, II and III) recovered(31.6%, 36.0%, 36.4%,) according to follicle size(<2 mm; 2 to 6 mm; >6 mm). Fertilization rate was 60.0%, 67.7%, 70.6% and 56.4% and the proportion of embryos/zygotes was 11.1%, 7.1%, 5.0% and 2.8% in 20~$25^{\circ}C$/BO, 30~35$^{\circ}C$/BO, 20~$25^{\circ}C$/TALP and 30~35$^{\circ}C$ /groups, respectively.

• Korean Journal of Animal Reproduction
• /
• v.17 no.3
• /
• pp.249-255
• /
• 1993

Canine follicular oocytes were used to establish a reliable system for maturation and fertilization in vitro. Ovaries were obtained from either slaughter house or hormone-primed bitches of mixed breeds. The oocytes were recovered by mincing the ovaries in M2＋BSA. Good quality of oocyte-cumulus complexes (OCCs) were selected and cultured in TCM 199 containing 15% fetal calf serum(FCS) for 24~56 h in an atmosphere of 5% CO2 at 39$^{\circ}C$. Maturation rate of follicular oocytes was >87% showing metaphase I. Unlike other domestic animals the cumulus expansion did not occur fully in canine OCCs although minimum expansion was found between the cumulus cells and corona radiata cells, the clear nuclear morphology was presented for the first time by rapid staining. The IVM system used in this study may be useful to obtain fully maturated metaphase I oocyte in dog.

• Reproductive and Developmental Biology
• /
• v.32 no.2
• /
• pp.71-79
• /
• 2008

Surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF MS) is one of the recently developed proteomic technologies which is based on capturing proteins and peptides by chemically modified surfaces and highly sensitive for the analysis of complex biological samples. In the present study, to gain insights into oocyte maturation and early embryo development, SELDI-TOF-MS was used to find the protein candidates that are specifically or prominently expressed in porcine oocytes at the in vitro matured metaphase II (MIIl) and germinal vesicle (GV) stages. By selected CM10 chip, 16 candidates were found to be up-regulated in GV stage oocytes compared with in MII stage oocytes, their molecular weights were 8,180 (2 candidates), 10,226 (5 candidates), 15,767 (5 candidates) and 16,770 (4 candidates) Da respectively. And the expression of 29 candidates were higher in MII than in GV stage oocytes, their molecular weight were 10,832 (3 candidates), 17,743 (8 candidates), 20,122 (3 candidates), 22,131 (3 candidates), 24,857 (7 candidates) and 33,507 (5 candidates) Da, respectively. The expression of selected 13 candidates (0.2 and 1.0 % error tolerances) were analyzed using real time RT-PCR. The proteins that differentially regulated during oocyte in vitro maturation in the pigs may be potential biomarkers of oocyte maturation and quality.

• Journal of Embryo Transfer
• /
• v.31 no.1
• /
• pp.9-12
• /
• 2016

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-specific growth factors that regulate many critical processes involved in early folliculogenesis and oocyte maturation. In this study, effects of GDF9 and BMP15 treatment during in vitro maturation of porcine oocytes upon development after parthenogenetic activation were investigated. Neither GDF, BMP15 alone nor in combination affects the number and viability of cumulus cells or the rates of oocyte maturation and blastocyst development. However, the treatment of GDF9 on porcine oocytes increased the number of trophectodermal (TE) cells of blastocysts derived from activated oocytes (P<0.05). The treatment of BMP15 increased the cell numbers of both inner cell mass (ICM) and TE cells (P<0.05). The treatment with the combination of GDF9 and BMP15 further increased the numbers of ICM and TE cells, compared with GDF9 or BMP15 treatment alone (P<0.05). In conclusion, the treatment of GDF9 or BMP15 (or both) enhanced the quality of blastocysts via the increased number of ICM and/or TE cells.

• Journal of Animal Reproduction and Biotechnology
• /
• v.34 no.2
• /
• pp.75-85
• /
• 2019

Omega-3 α-linolenic acid and omega-6 linoleic acid are essential fatty acids for health maintenance of human and animals because they are not synthesized in vivo. The purpose of this study was to evaluate the effect of α-linolenic acid and linoleic acid supplementation on in vitro maturation and developmental potential of porcine oocytes. Various concentrations of α-linolenic acid and linoleic acid were added into in vitro maturation medium, and we evaluated the degree of cumulus expansion, oocyte nuclear-maturation rate, blastocyst rate, blastocyst quality, and levels of prostaglandin E₂, 17β-estradiol, and progesterone in the spent medium. High doses (100 μM) of α-linolenic acid and linoleic acid supplementation significantly inhibited cumulus expansion and oocyte nuclear maturation, and prostaglandin E₂ synthesis also significantly decreased compared with other groups (p < 0.05). Supplementation of 50 μM α-linolenic acid and 10 μM linoleic acid showed higher quality blastocysts in terms of high cell numbers and low apoptosis when compared with other groups (p < 0.05), and synthesis ratio of 17β-estradiol / progesterone also significantly increased compared with control group (3.59 ± 0.22 vs. 2.97 ± 0.22, 3.4 ± 0.28 vs. 2.81 ± 0.19, respectively; p < 0.05). Our results indicated that supplementation with appropriate levels of α-linolenic acid and linoleic acid beneficially affects the change of hormone synthesis (in particular, an appropriate increase in the 17β-estradiol / progesterone synthesis ratio) for controlling oocyte maturation, leading to improved embryo quality. However, high doses of α-linolenic acid and linoleic acid treatment results in detrimental effects.

• Journal of Embryo Transfer
• /
• v.4 no.1
• /
• pp.46-55
• /
• 1989

The effects of an antiprogesterone (RU 486) and an antiestrogen (tamoxifen) on ovulatory response and oocyte morphology were examined in pregnant mare serum gonadotropin (PMSG)-primed immatare female rats (28 days of age): a comparison has been made on two different regirnens primed with a "control" dose (4 IU) and a "superovulatory" dose (40 IU) of PMSG. Females for control control regimen received three consecutive injections of lmg RU486, lmg tamoxifen, or vehicle at 24, 36 and 48hr, and were killed at 72l'r after PMSG. Animals for superovalatory regimen received lmg RU486, 2.5mg tamoxifen, or vehicle fouowlag the injection schedule comparable to control regimen, and were killed at 60 and 72hr after PMSG. Compared to vehicle group, there was a significant reduction in ovulatory response as judged by the proportion of rats ovulating andi or by the mean number of oocytes per rat for each treatment of RU486 and tamoxifen in both regimens. The activity of tamoxifen in inhibiting the ovulatory response was greater in control, but less in superovulatory regimen than that of RU486 based on the dose employed for each antisteroid. In both regimens, RU 486 did not have any effect 6n the changes in the proportion of degenerate oocytes as well as ovarian weight, well tamoxifen treatment resulted in a marked promotion of oocyte degeneration as well as a great reduction in ovarian weight, compared to each parameter of vehicle group. RU486 treatment in each regimen did not alter the serum levels of any steroid hormones observed. Howerver, tamoxifen treatment was associated with significant increases in serum 17$\beta$-estradiol and decreases in progesterone in both regimens; also significant increases in androgens in superovulatory regimen. The results illustrate the relative inhibitory activity of RU486 and tamoxifen indicating major steroid hormone involved in PMSG-induced ovulation: 17$\beta$-estradiol for control and progesterone for superovulatory regimen. It also appears that tamoxifen-associated elevation of circulating 17$\beta$-estradiol andi or androgens could be in part, a contributing factor to the promotion of oocyte degeneration presumably by producing a hostile oviductal environment after ovulation.ent after ovulation.

• Clinical and Experimental Reproductive Medicine
• /
• v.17 no.2
• /
• pp.115-121
• /
• 1990

Ultrasonically guided oocyte collection gradually replaces laparoscope in many IVF center. In present study, we compare the efficacy of both methods in our IVF program. Totally 377 cycles which were undertaken in vitro fertilization treatment were divided into 2 groups. Ultrasonically guided transvaginal follicular aspiration was performed in 188 cycles and laparoscopic follicular aspiration was performed in 189 cycles under local anesthesisa. The mean age for both groups was similar. Follicular recruitment was achieved with human menopausal gonadotropin (hMG) or a com bination of clomiphene citrate and hMG or a combination of FSH and hMG. In the ultrasonically guided aspiration group, 1821 follicles were aspirated with 61.8% of recovery rate (1125 oocytes), 81.5% of embryo transfer rate (145 cycles) and (17%), 26 cases intrauterine pregnancies were estabilished. In the laparoscopic group, 604 follicles were aspirated with 68.7% recovery rate (445 oocytes) and a 79.9% ET rate (127 cycles), 11 cases (8.7%) intrauterine pregnancies were estabilished. A valid comparison of these data is not possible because the 2 groups are dissimilar for factors known to influence oocyte development and recovery. No statistically significant differences could be demonstrated between 2 groups in all but the recovery rate and clinical pregnancy rate, In ultrasound group, the clinical pregnancy rate was significantly higher than that of laparoscope group. The potentially detrimental effect of CO2 pnemoperitonium present during laparoscope but not in ultrasound guided recovery on ova quality may underlie the observed difference in the clinical pregnancy rate between the 2 groups. Ultrasound guided aspiration seems to be as effective as laparoscopy in terms of oocyte retrieval and conception rate. Furthermore, the procedure is simple and inexpensive, it may replace laparoscopy as a method for oocyte collection in most patients who undergo IVF.