• Journal of Animal Reproduction and Biotechnology
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• v.36 no.4
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• pp.183-188
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• 2021

In vitro maturation (IVM) of oocytes is the procedure where the immature oocytes are cultivated in a laboratory until they are mature. Since IVM oocytes generally have low developmental competence as compared to those matured in vivo, development of an optimal IVM culture system by fine-tuning culture conditions is crucial to maintain high quality. In-depth knowledge and a deep understanding of the in vivo physiology of oocyte maturation are pre-requisites to accomplish this. Within ovarian follicles, various signaling pathways that drive oocyte development and maturation regulate interaction between oocytes and surrounding somatic cells. This review discusses the sonic hedgehog (SHH) signaling pathway, which has been demonstrated to be intimately involved in folliculogenesis and oocyte maturation. Advances in elucidating the role of the SHH signaling pathway in oocyte maturation will aid attempts to improve the current inferior in vitro oocyte maturation system.

• Asian-Australasian Journal of Animal Sciences
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• v.11 no.5
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• pp.491-497
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• 1998

Four types of serum supplements viz. estrus cow serum (ECS), estrus buffalo serum (EBS), pro-estrus buffalo serum (PrBS) and post-estrus buffalo serum (PtBS), added to TCM-199, were evaluated for in vitro maturation and fertilization of buffalo follicular oocytes. The oocytes were recovered from buffalo ovaries after slaughter, using either aspiration or scoring (multiple incisions) method. The recovered oocytes were categorized as A, B and C based on their cumulus investment and ooplasm homogeneity and cultured in four media. The in vitro matured oocytes were inseminated with $1{\times}10^6$ spermatozoa washed in 2.9% sodium citrate solution. The scoring method yielded greater number of morphologically good oocytes than the aspiration method (3.85 vs 1.76 per ovary, p < 0.01). The maturation rates of three categories of oocytes did not differ from one another. The maturation rates of 80.00, 82.08, 78.77 and 66.23%, while the fertilization rates of 54.54, 55.38, 52.80 and 36.76% were recorded for media containing ECS, EBS, PrBS, and PtBS, respectively. The medium containing PtBS gave lower maturation, as well as fertilization, rates than the other three media (p < 0.05). Thus, the scoring method was better than the aspiration method for the recovery of follicular oocytes. The oocytes categorized A, B and C had similar maturation capabilities. The TCM-199 containing buffalo/cow serum collected at pro-estrus or estrus appeared better for in vitro maturation and fertilization of buffalo follicular oocytes than that containing serum collected at post estrus.

• Journal of Embryo Transfer
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• v.9 no.1
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• pp.1-6
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• 1994

This experiment was investigated the effect of presence of granulosa cells from follicles of different size on bovine oocyte maturation, cleavage and development to late stage. The nuclear and cytoplasmic maturation of oocytes in the IVM-IVF system are critical for subsequent embryo development. Granulosa cells when the co-cultured with oocytes may interact with cumulus-oocytes complexes and influence the development competence of the oocytes. Granulosa cells from medium (2~6 mm) and large(>1O mm) size follicles were recovered by aspiration, washed 3 times by centrifugation at 500 x g for 5 min. and used for co-culture at a concentration of 2~3 x 106 cells/mi. The oocytes were matured in vitro (IVM) for 24 hrs. in TCM-199 supplemented with 35 $\mu$g/ml FSH, 10 $\mu$g/ml LH, 1 $\mu$g/ml estradiol-17$\beta$ and granulosa cells at 39$^{\circ}C$ under 5% $CO_2$ in air. They were fertilized in vitro (IVF) by epididymal spermatozoa treated with heparin for 24 hrs., and then the zygotes were co-cultured in vitro (I VC) with bovine oviductal epithelial cells for 7 to 9 days. The assessment of maturation revealed that Grade J oocytes showed significantly(P

• Journal of Embryo Transfer
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• v.23 no.2
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• pp.77-80
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• 2008

In the present study, effects of concentration of cryoprotectant solutions on the nuclear maturation of vitrified-thawed porcine oocytes were examined. Also, the developmental capacity of vitrified-thawed immature porcine oocytes following ICSI was investigated. Oocytes were cultured in NCSU-23 medium supplemented with 5% FBS at $38^{\circ}C$ in 5% $CO_2$ and air. The in vitro maturation rate of vitrified-thawed oocytes ($24.1{\pm}2.5%$) was lower than that of the control ($46.0{\pm}3.2%$, p<0.05). The in vitro maturation rate of vitrified-thawed oocytes treated with $1.0{\sim}5.0\;ug$ CB + NCSU- 23 medium were $22.2{\pm}3.0%$, $30.7{\pm}3.2$, $46.3{\pm}3.1%$, $38.5{\pm}3.2%$, respectively. The in vitro maturation rate ($46.3{\pm}3.4%$) of the vitrified-thawed oocytes treated with $3.0\;{\mu}g$ CB for 30 min was the highest of all vitrification groups. When the in vitro developmental rates of the vitrified-thawed (with EDS and EDT) oocytes following ICSI were $18.5{\pm}2.5%$, $16.4{\pm}2.1%$, respectively. This results were lower than the control group ($24.0{\pm}2.5%$).

• Journal of Embryo Transfer
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• v.12 no.3
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• pp.293-300
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• 1997

Supplementation of maturation medium with additional granulosa cells has beneficial effect on in vitro maturation of bovine follicular oocytes and their subsequent cleavage and development in vitro. However, maturation system using granulosa cells have some disadvantages that collection of granulosa cells is cumbersome and metabolic activity of the cells is variable according to ovarian cycle or follicular size. We hypothesized that bovine immsture oocytes matured without granulosa cell coculture can fertilize and develop normally if the medium volume per oocyte is reduced during in vitro maturation. Immature oocytes were matured for 24 hours in a TCM199 containing 10% fetal calf serum, anterior pitultary hormone (0.02 AU /ml Antrinⓡ) and estradiol with or without granulosa cells in vitro. In Group 1, 35 to 40 oocytes were matured in a well of 4-well plastic dish containing 500 $\mu$l of maturation medium and granulosa cells, and 9 to 10 oocytes were matured in a 50-$\mu$l drop of maturation medium without granulosa cells in Group 2. After maturation, oocytes were coincubated with sperm for 30 hours in a modified Tyrode's medium (IVF). Inseminated oocytes were cultured in a microdrop (30 $\mu$l) of a synthetic oviduct fluld medium (SOFM) containing BSA, Minimum Essential Medium essential and non-essential amino acids for 9 days. As a preliminary experiment, we investigated the beneficial effect of granulosa cells during maturation on subsequent cleavage and development using the same type of culturedishes (4-well dish). Granulosa cells could not increase embryo cleavage after fertilization but significantly improved (p<0.05) embryo development to expanding blastocyst (Table1 and 2). In Group 1, 68 and 80% of inseminated oocytes have cleaved at 30 hours and 2 days after IVF, respectively, which is similar (p>0.05) to the result of Group 2 (69% at 30 hours and 78% at 2 days after IVF). The oocytes in Group 2 showed 21 and 11% of developmental rates to expanding and hatching blastocysts, respectively, which was not significantly different (p>0.05) from those (20 and 10%, respectively) of oocytes in Group 1. In conclusion, it has been clarified that a microdrop culture system without granulosa cells for in vitro maturation can support bovine embryonic development to blastocyst in vitro as readily as a granulosa cell coculture system.

• Asian-Australasian Journal of Animal Sciences
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• v.8 no.2
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• pp.123-127
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• 1995

In vitro embryo production (IVP) is affected by various factors during in vitro maturation, fertilization, and development. In this experiment, the effect of ovary type, quality of follicular oocyte, medium used for fertilization, presence of hormone in medium, sperm concentration on in vitro maturation and fertilization were examined for effective IVP. In vitro maturation was carried out using TCM-199 supplemented with 15% FCS and hormones in 5% $CO_2$ incubator for 24h. In vitro fertilization was performed with frozen-thawed sperm in modified mTALP medium containing 0.3% BSA, $10{\mu}g/ml$ heparin, and 5mM/ml caffeine for 24h. The fertilized embryos were co-cultured on monolayer of cumulus cells in TCM-199. When oocytes were collected from functionally active and inactive ovaries, maturation rate was 76.9 and 7.7%, respectively. When oocytes were classified morphologically to good and poor grades, maturation rate was 75 and 58.8%, respectively. FSH + LH + $E_2$ (86.4%) showed higher maturation rate than control (53.0%) and FSH (73%). The fertilization rate was 28.2, 100 and 91.7% in $1.6{\times}10^5$, $5.0{\times}10^5$ and $10.0{\times}10^5$ sperm concentration per ml. When oocytes were fertilized in mTALP and BO media, fertilization and cleavage rates of oocytes in mTALP were higher (84.3 and 56.9%) than those (67.4 and 23.3%) in BO medium. In this experiment, in vitro maturation, fertilization and development of oocytes were affected by type of ovary, grade of oocyte, hormones, sperm concentration and fertilization medium.

• Korean Journal of Animal Reproduction
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• v.19 no.4
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• pp.251-258
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• 1996

These studies were carried out to investigate the effect of gonadotropins (GTH), fetal calf serum (FCS), porcine follicular fluid (pFF) and FCS and pFF fractions obtained by the gel filtration on in vitro maturation of porcine follicular fluid. When the oocytes were cultured in TCM-199, the maturation rate was higher in pFF than in FCS in both with or without GTH and in pFF the maturation rate was higher in with GTH than in without GTH. In case of without GTH, pFF increased maturation rates in TCM-199, but not in Whitten's medium (WM). When the oocytes were cultured in WM supplemented with FCS fractions, the maturation rate(51.6%) of oocytes was significantly (P<0.05) higher in fraction B (about 30∼70 kDa) than in control, FCS and other fractions. When oocytes were cultured in WM supplemented with pFF fractions, fractions B (about 30∼70 kDa) and D (about 1∼10 kDa) were significantly (P<0.05) higher than in control, pFF and other fractions. In conclusiion, the addition of gonadotropins into the maturation media was effective for oocyte maturation. The addition of pFF was more effective than addition of FCS for maturation of porcine oocytes in vitro. And fraction B from FCS and fractions B and D from pFF was effective for oocyte maturation.

• Korean Journal of Animal Reproduction
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• v.22 no.1
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• pp.95-99
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• 1998

Three experiments were conducted with follicular oocytes, to compare some somatic cells, growth factors and media for in vitro maturation of bovine oocytes. In the first experiment, the type of somatic cells had no effects on in vitro maturation of bovine follicular ooctyes. In the second experiment, oocytes were matured in TCM199 su, pp.emented with growth factors on IVM of bovine follicular oocytes, then all were co-cultured with cumulus cells. The proportion of used oocytes that developed to expanding blastocysts was 22.2%, 20.2%, 17.7%, 22.2%, 24.4% and 20.2% after maturation in TCM199 su, pp.emented with control, insulin, IGF-I, IGF-Ⅱ, FGF and EGF, respectively. In the third experiment, oocytes were matured in BO, Ham's F10 and TCM199, then all were fertilized in BO, and embryos cultured in BO, Ham's F10 and TCM199, respectively. Cleavage rates in BO were 90%, had higher than in Ham's F10(80%) or in TCM199(64%). But production of expanding blastocysts in TCM199(21%) or Ham's F10(20.6%), had higher than in BO(4.6%).

• Korean Journal of Animal Reproduction
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• v.12 no.2
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• pp.120-124
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• 1988

Rat oocyte-cumulus complexes were cultured in verious media in order to induce maturation division in vitro. When the complexes were cultured in mKRB containing estrous rat serum (ERS) and FCS of 5% instead of BSA higher proportions(83.3 and 86.7%) of oocytes matured to metaphase II in 20h compared to control(75%) and t도 maturation rates in mKRB plus FCS were generally higher than those in mKRB plus ERS. Fertilization and early cleavage rates in vitro of the intact oocytes matured in mKRB containing BSA and FCS were generally higher than those of cumulus-removed oocytes and these rates were higher in mKRB containing 5% FCS than those in mKRB containing BSA. These results indicate that maturation rate in vitro was greatly increased by the addition of FCS instead of BSA to mKRB solution and the presence of cumulus cells around oocytes prior to sperm insemintion may be responsible for the increase of in vitro fertilization and early cleavage rates.

• Reproductive and Developmental Biology
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• v.33 no.3
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• pp.125-131
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• 2009

The aim of this study is to investigate the effect of porcine epididymal fluid (pEF) on in vitro-maturation and subsequent fertilization of porcine follicular oocytes. Porcine cumulus-oocytes complexes retrieved from antral follicles were cultured in tissue culture medium (TCM)-l99 supplemented with pEF of different concentrations. At 48 h after culture, development of oocytes to germinal vesicle (GV) breakdown, metaphase I, anaphase-telophase I, and metaphase II were examined Significant (p<0.05) increase in the proportion of oocytes developed to MII stage was observed in oocytes cultured in pEF-containing TCM-l99 than in oocytes cultured in pEF-free TCM-199 (46.2% vs 16.7%), which was a dose-dependent manner. Subsequently, the proportion of monospermic fertilization were significantly (p<0.05) increased in oocytes cultured in the TCM supplemented with pEF than those cultured in pEF-free TCM-199 (51.0% vs 24.1%). In the second series of experiment, the percentage of MII oocytes was significantly (p<0.05) increased after exposure of oocytes to pEF during the first 22 h period of culture than after exposure of oocytes to pEF during the next 24 h of culture, while no significant difference in the percentage of monospermy was observed. The results of this study demonstrate that pEF contains at least enhancing component(s) for nuclear maturation.