The role of sonic hedgehog signaling pathway in in vitro oocyte maturation

Sanghoon Lee,Jongki Cho 한국동물생명공학회(구 한국동물번식학회) 2021 Journal of Animal Reproduction and Biotechnology Vol.36 No.4

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.

Epidermal Growth Factor(EGF)와 Transforming Growth Factor-$\alpha$(TGF-$\alpha$)가 돼지 난포란의 체외성숙에 미치는 영향

임정훈,박병권,이규승 한국동물번식학회 1997 Reproductive & developmental biology Vol.21 No.2

The present study examined the effects of epidermal growth factor(EGF) and transforming growth factor-$\alpha$(TGF-$\alpha$) on in vitro maturation of porcine follicular oocytes. Basic medium used TCM-HEPES, and oocytes cultured for 42 hours in vitro. The results obtained are as follows; 1. The nuclear maturation rates of EGF-treated groups(10ng/ml, 75.9% ; 30ng/ml, 69.2% ; 50ng/ml, 67.2% ; 100ng/ml, 71.0%) on the porcine oocytes cultured in medium without pFF in vitro were significantly(P<0.01) higher than those of non-treated group(57.1%). When the oocytes were cultured in media with 10%(v/v) pFF, the nuclear maturation rates of 30ng EGF/ml(77.1%) treated group were significantly(P<0.01) higher than those of non-(59.2%) and EGF-treated groups(10ng/ml, 65.4% ; 50ng/ml, 65.5% ; 100ng/ml, 70.4%). 2. The nuclear maturation rates of 30ng TGF-$\alpha$/ml treated group(71.9%) in media without pFF in vitro were significatnly(P<0.01) higher than those of non-(57.1%) and TGF-$\alpha$ treated groups(10ng/ml, 60.4% ; 50ng/ml, 65.4% ; 100ng/ml, 60.0%). When the oocytes were cultured in media with 10%(v/v) pFF, the nuclear maturation rates of 30 and 50ng TGF-$\alpha$/ml(77.4% and 79.6%) treated groups(10ng/ml, 64.2% ; 100ng/ml, 61.6%). 3. On the effect of EGF(30ng/ml) and/or TGF-$\alpha$(30ng/ml) treated groups in medium without pFF in vitro, the nuclear maturation rates indicated 57.3, 60.4, 75.9 and 79.7% in media with no EGF & TFG-$\alpha$, TGF-$\alpha$ only, EGF only nad EGF+TGF-$\alpha$ treated groups, respectively. The nuclear maturation rates in medium with EGF only or EGF+TGF-$\alpha$ were significantly(P<0.01) higher than those non- and TGF-$\alpha$ treated groups. When the oocytes were cultured in media with 10%(v/v) pFF, the nuclear maturation ratesof EGF+TGF-$\alpha$ treated group(75.9%) were significantly(P<0.01) higher than those of non-(59.2%), TGF-$\alpha$ only (64.2%) and EGF only(69.4%) treated groups.

Development and pregnancy rates of Camelus dromedarius-cloned embryos derived from in vivo- and in vitro-matured oocytes

Son Young-Bum,Jeong Yeon Ik,Jeong Yeon Woo,Olsson Per Olof,Hossein Mohammad Shamim,Cai Lian,Kim Sun,Choi Eun Ji,Sakaguchi Kenichiro,Tinson Alex,Singh Kuhad Kuldip,Rajesh Singh,Noura Al Shamsi,Hwang Wo 아세아·태평양축산학회 2022 Animal Bioscience Vol.35 No.2

Objective: The present study evaluated the efficiency of embryo development and pregnancy of somatic cell nuclear transfer (SCNT) embryos using different source-matured oocytes in Camelus dromedarius. Methods: Camelus dromedarius embryos were produced by SCNT using in vivo- and in vitro- matured oocytes. In vitro embryo developmental capacity of reconstructed embryos was evaluated. To confirm the efficiency of pregnancy and live birth rates, a total of 72 blastocysts using in vitro- matured oocytes transferred into 45 surrogates and 95 blastocysts using in vivo- matured oocytes were transferred into 62 surrogates by transvaginal method. Results: The collected oocytes derived from ovum pick up showed higher maturation potential into metaphase II oocytes than oocytes from the slaughterhouse. The competence of cleavage, and blastocyst were also significantly higher in in vivo- matured oocytes than in vitro- matured oocytes. After embryo transfer, 11 pregnant and 10 live births were confirmed in in vivo- matured oocytes group, and 2 pregnant and 1 live birth were confirmed in in vitro- matured oocytes group. Furthermore, blastocysts produced by in vivo-matured oocytes resulted in significantly higher early pregnancy and live birth rates than in vitromatured oocytes. Conclusion: In this study, SCNT embryos using in vivo- and in vitro-matured camel oocytes were successfully developed, and pregnancy was established in recipient camels. We also confirmed that in vivo-matured oocytes improved the development of embryos and the pregnancy capacity using the blastocyst embryo transfer method. Objective: The present study evaluated the efficiency of embryo development and pregnancy of somatic cell nuclear transfer (SCNT) embryos using different source-matured oocytes in Camelus dromedarius.Methods: Camelus dromedarius embryos were produced by SCNT using in vivo- and in vitro- matured oocytes. In vitro embryo developmental capacity of reconstructed embryos was evaluated. To confirm the efficiency of pregnancy and live birth rates, a total of 72 blastocysts using in vitro- matured oocytes transferred into 45 surrogates and 95 blastocysts using in vivo- matured oocytes were transferred into 62 surrogates by transvaginal method.Results: The collected oocytes derived from ovum pick up showed higher maturation potential into metaphase II oocytes than oocytes from the slaughterhouse. The competence of cleavage, and blastocyst were also significantly higher in in vivo- matured oocytes than in vitro- matured oocytes. After embryo transfer, 11 pregnant and 10 live births were confirmed in in vivo- matured oocytes group, and 2 pregnant and 1 live birth were confirmed in in vitro- matured oocytes group. Furthermore, blastocysts produced by in vivo-matured oocytes resulted in significantly higher early pregnancy and live birth rates than in vitromatured oocytes.Conclusion: In this study, SCNT embryos using in vivo- and in vitro-matured camel oocytes were successfully developed, and pregnancy was established in recipient camels. We also confirmed that in vivo-matured oocytes improved the development of embryos and the pregnancy capacity using the blastocyst embryo transfer method.

제주말에서 난포 크기에 따른 난포란의 체외성숙

류재규,강태영 한국임상수의학회 2010 한국임상수의학회지 Vol.27 No.1

In this study, we investigated the number of follicles, oocyte recovery rate and oocyte competence after in vitro maturation according to the size of follicle. And equine oocyte competence after in vitro maturation was investigated in terms of the diameter of follicle with criteria of maturation: nuclear stage after Hoechst staining. The average number of follicles per ovary with middle size (11-20 mm, 2.68) was higher than those of small (5-10 mm,0.74) and large size follicle (> 21 mm, 1.63), therefore medium follicle (53.1%) had higher proportion than other size of follicles. The average numbers of follicle per ovary was 5.05. The rate of oocyte recovery in small (54.5%) and middle follicle (50%) was higher than that in large follicle (40.9%). After culture for 48 h in Medium 199, 50%,45.5%, and 44.4% of oocytes from the follicles with diameters of 5-10, 11-20, > 21 mm, respectively reached the metaphase II stage. This is the first report showing number of follicle, oocyte recovery rate according to follicular size, and in vitro oocyte maturation in Jeju mare in Korea. To fulfill in vitro equine embryo production, further studies such as the seasonal effect, in vitro fertilization etc is need.

Effect of Porcine Epididymal Fluid on In Vitro Maturation of Porcine Germinal Vesicle Oocyte

Yim, Cha-Ok,Kim, Kyoung-Woon,Kim, Byung-Ki The Korean Society of Animal Reproduction 2008 Reproductive & developmental biology Vol.32 No.3

The aim of this study was to investigate what components of porcine epididymal fluid (pEF) influences the nuclear maturation of porcine germinal vesicle oocytes. Porcine cumulus-oocytes complexes from follicles were cultured in TCM 199 containing pEF. After 48 h cultures, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. Maturation rate of oocytes was significantly increased in media supplemented with 10% pEF during in vitro maturation (IVM) than in those without pEF. When lipid component of pEF was removed by treating n-heptane, no significant difference was observed in maturation of oocytes between n-heptane treatrment and intact pEF group. However, the proportion of oocytes reaching at metaphase II (M II) was significantly (p<0.05) decreased in the oocytes cultured in media containing trypsin-treated pEF compared to those in media with intact pEF. When porcine GV oocytes were matured in the medium supplemented with intact pEF or pEF heated at $56^{\circ}C$ and $97^{\circ}C$, rates of oocytes remained at GV stage were 11.7%, 29.4% and 42.0%, respectively. However, there were no difference in proportion of oocytes reaching at MII stage among intact pEF group and $56^{\circ}C$ group. Present study suggests that 1) pEF contains an enhancing component(s) for nuclear maturation in vitro of oocytes, 2) protein(s) of pEF may be capable to promote nuclear maturation in vitro, and 3) enhancing component for nuclear maturation may consist of two factors, which are responsible for germinal vesicle breakdown (GVBD) and promotion of MII stage.

Effect of Porcine Epididymal Fluid on In Vitro Maturation of Porcine Germinal Vesicle Oocyte

Cha Ok Yim,Kyoung Woon Kim,Byung Ki Kim 한국동물생명공학회(구 한국동물번식학회) 2008 Reproductive & developmental biology Vol.32 No.3

The aim of this study was to investigate what components of porcine epididymal fluid (pEF) influences the nuclear maturation of porcine germinal vesicle oocytes. Porcine cumulus-oocytes complexes from follicles were cultured in TCM 199 containing pEF. After 48 h cultures, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. Maturation rate of oocytes was significantly increased in media supplemented with 10% pEF during in vitro maturation (IVM) than in those without pEF. When lipid component of pEF was removed by treating n-heptane, no significant difference was observed in maturation of oocytes between n-heptane treatrment and intact pEF group. However, the proportion of oocytes reaching at metaphase II (M II) was significantly (p<0.05) decreased in the oocytes cultured in media containing trypsin-treated pEF compared to those in media with intact pEF. When porcine GV oocytes were matured in the medium supplemented with intact pEF or pEF heated at 56'C and 97'C, rates of oocytes remained at GV stage were 11.7%, 29.4% and 42.0%, respectively. However, there were no difference in proportion of oocytes reaching at MII stage among intact pEF group and 56'C group. Present study suggests that 1) pEF contains an enhancing component(s) for nuclear maturation in vitro of oocytes, 2) protein(s) of pEF may be capable to promote nuclear maturation in vitro, and 3) enhancing component for nuclear maturation may consist of two factors, which are responsible for germinal vesicle breakdown (GVBD) and promotion of MII stage.

Expression of Transcripts in Marmoset Oocytes Retrieved during Follicle Isolation Without Gonadotropin Induction

Kim, Yoon Young,Kang, Byeong-Cheol,Yun, Jun Won,Ahn, Jae Hun,Kim, Yong Jin,Kim, Hoon,Rosenwaks, Zev,Ku, Seung-Yup MDPI 2019 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.20 No.5

<P>The in vitro maturation of oocytes is frequently used as an assisted reproductive technique (ART), and has been successfully established in humans and rodents. To overcome the limitations of ART, novel procedures for the in vitro maturation of early follicles are emerging. During the follicle isolation procedure, the unintended rupture of each follicle leads to a release of extra oocytes. Such oocytes, which are obtained during follicle isolation from marmosets, can be used for early maturation studies. Marmoset (<I>Callithrix jacchus</I>), which is classified as a new-world monkey, is a novel model that has been employed in reproductive biomedical research, as its reproductive physiology is similar to that of humans in several aspects. The ovaries of female marmosets were collected, and the excess oocytes present during follicle isolation were retrieved without pre-gonadotropin induction. Each oocyte was matured in vitro for 48 h in the presence of various concentrations of human chorionic gonadotropin (hCG) and epidermal growth factor (EGF), and the maturity of oocytes and optimal maturation conditions were evaluated. Each oocyte was individually reverse-transcribed, and the expression of mRNAs and microRNAs (miRs) were analyzed. Concentrations of hCG significantly affected the maturation rate of oocytes [the number of metaphase II (MII) oocytes]. The expression of <I>BMP15</I> and <I>ZP1</I> was highest when the oocytes were matured using 100 IU/L of hCG without pre-treatment with gonadotropins, and that of <I>Cja-mir-27a</I> was highest when cultured with follicle stimulating hormone. These results suggest that these up-regulated miRs affect the maturation of oocytes. Interactions with other protein networks were analyzed, and a strong association of BMP15 and ZP1 with sperm binding receptor (ACR), anti-Müllerian hormone (AMH), and AMH receptor was demonstrated, which is related to the proliferation of granulosa cells. Collectively, on the basis of these results, the authors propose optimal maturation conditions of excess oocytes of marmoset without in vivo gonadotropin treatment, and demonstrated the roles of miRs in early oocyte maturation at the single-cell level in marmosets.</P>

Effect of Co-Culture with Mammalian Spermatozoa on In Vitro Maturation of Porcine Cumulus-Enclosed Germinal Vesicle Oocytes

Kim Byung Ki 한국동물생명공학회(구 한국동물번식학회) 2004 Reproductive & developmental biology Vol.28 No.4

In vitro maturation of denuded porcine immature oocytes can be enhanced by co-incubation with spermatozoa even before fertilization. This study was to determine whether the addition of spermatozoa into the culture medium could influence the nuclear maturation of porcine cumulus-enclosed germinal vesicle (GV) oocytes. Cumulus-oocyte complexes (COCs) were collected from follicles of 3- to 5-mm diameter. Porcine COCs were cultured in tissue culture medium containing spermatozoa. After 48 h culture, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. The proportion of oocytes reaching at metaphase II was significantly (P < 0.05) increased in the oocytes cultured in media containing spermatozoa compared to those in media without spermatozoa (52.3% vs 12.5%). No difference in the percentage of metaphase II was observed among the different periods of spermatozoa exposure and among the spermatozoa from different species. The proportion of oocytes reaching metaphase II was significantly different between high and low concentrations of spermatozoa. The present study suggests that manunalian spermatozoa contain a substance(s) that improves nuclear in vitro maturation of porcine cumulus-enclosed GV oocytes. Enhancing effect of spermatozoa for in vitro maturation of oocytes is a highly dose-dependent.

Effects of Follicle Stimulating Hormone and Human Chorionic Gonadotrophin on the In Vitrto Maturation of Canine Oocytes

Min Kyu Kim,Hyun Ju OH,Goo Jang,So Gun Hong,Jung Eun Park,Hye Jin Kim,Hyung Suk Lee,Sang Cheol Kim,Sung Keun Kang,Byeong Chun Lee 한국동물생명공학회(구 한국동물번식학회) 2007 Reproductive & developmental biology Vol.31 No.1

The present study investigated the effects of follicle stimulating hormone (FSH) and human chorionic gonadotrophin (hCG) on the nuclear maturation of canine oocytes. Oocytes were recovered from mongrel female ovaries in various reproductive states; follicular, luteal or anestrous stage. Oocytes were cultured in serum-free tissue culture medium (TCM)-199 supplemented with various concentrations of FSH (Exp. 1: 0, 0.5, 1.0 or 10 IU) or hCG (Exp. 2: 0, 0.5, 1.0 or 10 IU) or both (Exp. 3: 1 IU FSH + 1 IU hCG) for 72 hr to determine the effective concentration of these hormones, and to examine their combined effect. After maturation culture, oocytes were denuded in PBS containing 0.1% (w/v) hyaluronidase by gentle pipetting. The denuded oocytes were stained with 1.9 μM. Hoechst 33342 in glycerol and the nuclear state of oocytes was evaluated under UV light. More (p<0.05) oocytes matured to MII stage when follicular stage oocytes were supplemented with 1 IU FSH (6.2%) compared with the control, 0.1 or 10.0 IU FSH (0 to 1.2%). Significantly higher (p<0.05) maturation rate to MII stage was observed in follicular stage oocytes supplemented with 1.0 IU hCG (7.2%) compared with the control or other hCG supplemented groups (0 to 1.5%). However, the combination of FSH and hCG did not improve the nuclear maturation rate of canine oocyte (2.4 %) compared with FSH (6.2%) and hCG alone (7.2%). In conclusion, FSH or hCG alone significantly increased the maturation of canine oocytes to MII stage.

Use of immature oocytes in infertility treatment

Byung Chul Jee 한국발생생물학회 2010 한국발생생물학회 학술발표대회 Vol.29 No.-

Since the first case of pregnancy by in vitro matured oocyte was reported (Cha et al., 1991), in vitro maturation (IVM) could be used as an alternative choice for the treatment of infertile women with polycystic ovary syndrome (PCOS) and poor responders to ovarian stimulation and as one of the strategies for fertility preservation (Chian, 2004). Immature oocyte retrieval followed by IVM is a promising potential treatment option, especially for women who are infertile through PCOS. Although the pregnancy and implantation rates of IVM treatment are not as high as conventional IVF treatment, IVM treatment has many advantages for infertile women with PCOS, because this group of patients is extremely sensitive to stimulation with exogenous gonadotropins and is at increased risk of developing ovarian hyperstimulation syndrome (OHSS). Different protocols have been used before immature oocyte retrieval, indicating that there are beneficial effects with FSH or LH priming on oocyte maturation. To date, the clinical pregnancy and implantation rates obtained from IVM treatment in infertile women with PCOS are approximately 30-35% and 10-15% respectively (Chian, 2004). The clinical outcome has substantially improved in recent years with pregnancy rates between 20 and 54% and the postnatal follow-up studies of the children have been reassuring (Suikkari, 2008). Currently, more than 400 healthy infants have been reported with IVM method (Jurema and Nogueira, 2006; Suikkari, 2008). Although good results have been reported by some clinics, IVM has not yet become a mainstream fertility treatment. The most important reason for this is the lower chance of a live birth per treatment compared with conventional IVF. Despite its clinical success, there has been little information about the suitable conditions for human IVM. Therefore, improving developmental competency of immature oocytes continues to be an important concern of most IVM centers. Among many factors which affect efficacy of IVM, culture conditions are believed to be the most important factor, because different culture medium with changes of constituents can affect the oocyte maturation potential and subsequent embryonic development (Trounson et al., 2001). Currently, many different types of commercially available maturation media have been used in clinical IVM. They are commonly supplemented with hormone (recombinant FSH, hCG) and protein sources. Protein component may serve as a nitrogen source and act as a chelator of toxic metal ions and an antioxidant within culture media. In this respect, a development of well defined maturation medium supplemented with an efficient and safe protein source would improve IVM results. We previously reported that developmental competency of immature oocytes (either GV or MI) obtained from stimulated IVF cycles was comparable when matured in vitro with commercial G2 media supplemented by either human follicular fluids (hFF) or human serum albumin (HSA) (Jee et al., 2008). Our results suggest that hFF as a protein supplement for human in vitro maturation can be replaced by highly defined HSA. A development of well defined maturation medium should be continued in the effort to improve IVM results. More research is also needed to determine the roles of specific components and optimal culture conditions required in maturing oocytes. IVM of human oocytes retrieved from antral ovarian follicles is an emerging procedure quickly being incorporated into the realm of assisted reproductive technologies (ART). This new technology has several potential advantages over traditional controlled ovarian hyperstimulation for IVF, such as reduction of costs by minimizing gonadotropin and GnRH analogue use, elimination of OHSS, and simplicity of protocol. IVM of oocytes for ART in human beings still is undergoing refinement but currently is providing efficacy and safety outcome comparable to that of traditional IVF in recent selected studies. Implementing IVM into an established IVF practice is feasible and requires only a few simple adjustments. Crucial to the advancement and optimization of the technology is a better understanding of how to maximize immature oocyte developmental competence and endometrial receptivity.