Effect of oocyte chromatin status in porcine follicles on the embryo development in vitro

이주빈,이민구,Tao Lin,신현영,이재은,강정원,진동일 아세아·태평양축산학회 2019 Animal Bioscience Vol.32 No.7

Objective: The main goal of this study was to provide a morphological indicator that could be used to select high-quality oocytes of appropriate meiotic and developmental capabilities in pig. The higher quality of immature oocytes, the higher success rates of in vitro maturation (IVM) and in vitro fertilization (IVF). Thus, prior to the IVM culture, it is important to characterize oocytes morphologically and biochemically in order to assess their quality. Two of the largest indicators of oocyte quality are the presence of cumulus cells and status of chromatin. To investigate the effects of porcine oocyte chromatin configurations on the developmental capacity of blastocysts, we assessed oocyte chromatin status according to follicle size and measured the developmental potency of blastocysts. Methods: To sort by follicle size, we divided the oocytes into three groups (less than 1 mm, 1 to 3 mm, and more than 3 mm in diameter). To assess chromatin configuration, the oocytes were assessed for their stages (surrounded nucleolus [SN] germinal vesicle [GV], non-surrounded nucleolus [NSN] GV, GV breakdown, metaphase I [MI], pro-metaphase II [proMII], and metaphase II [MII]) at different maturation times (22, 44, and 66 h). To assess the development rate, oocytes of each follicle size were subjected to parthenogenetic activation for further development. Finally, GV oocytes were grouped by their chromatin configuration (SN, SN/NSN, and NSN) and their global transcriptional levels were measured. Results: SN GV oocytes were more suitable for IVF than NSN GV oocytes. Moreover, oocytes collected from the larger follicles had a greater distribution of SN GV oocytes and a higher developmental capacity during IVM, reaching MII more quickly and developing more often to blastocysts. Conclusion: Porcine oocytes with high-level meiotic and developmental capacity were identified by analyzing the relationship between follicle size and chromatin configuration. The porcine oocytes from large follicles had a significantly higher SN status in which the transcription level was low and could be better in the degree of meiotic progression and developmental capacity.

Endoplasmic Stress Inhibition during Oocyte Maturation Improves Preimplantation Development of Cloned Pig Embryos

Elahi, Fazle,Shin, Hyeji,Lee, Joohyeong,Lee, Eunsong The Korean Society of Embryo Transfer 2017 한국동물생명공학회지 Vol.32 No.4

Mitochondrial dysfunction is found in oocytes and transmitted to offspring due to maternal obesity. Treatment of obese mothers with endoplasmic reticulum (ER) stress inhibitors such as salubrinal (SAL) can reverse the mitochondrial dysfunction and result in normal embryonic development. Pig oocytes have also shown ER stress mostly in metaphase II stage. ER stress in oocytes may hinder the in vitro production of pig embryos. This study investigated the effect of ER stress inhibition by SAL treatment during in vitro maturation (IVM) of porcine oocytes at 1, 10, 50 and 100 nM concentrations. Firstly, we tested various concentrations of SAL. SAL at 10 nM showed higher (P < 0.05) developmental competence to the blastocyst stage (55.6%) after parthenogenesis (PA) than control (44.2%) while not different from other concentrations (49.2, 51.6, and 50.8% for 1, 50, and 100 nM, respectively). Secondly, we performed time-dependent treatment at 10 nM of SAL for IVM of oocytes. It revealed that treatment with SAL during 22 to 44 h of IVM significantly improved PA embryonic development to the blastocyst stage compared to control (40.5, 46.3, 51.7 and 60.2% for control, 0 to 22 h, 22 to 44 h and 0 to 44 h of IVM, respectively, P < 0.05). Glutathione (GSH) content is an indicator of cytoplasmic maturation of oocytes. Reactive oxygen species (ROS) have a harmful effect on developmental competence of oocytes. For this, we determined the intraoocyte levels of GSH and ROS after 44 h of IVM. It was found that SAL increased intraoocyte GSH level and also decreased ROS level (P < 0.05). Finally, we performed somatic cell nuclear transfer (SCNT) after treating oocytes with 10 nM SAL during IVM. SAL treatment significantly improved blastocyst formation of SCNT embryos compared to control (39.6% vs. 24.7%, P < 0.05). Our results indicate that treatment of pig oocytes with ER stress inhibitor SAL during IVM improves preimplantation development PA and cloned pig embryos by influencing cytoplasmic maturation in terms of increased GSH content and decreased ROS level in IVM pig oocytes.

MicroRNAs transfected into granulosa cells may regulate oocyte meiotic competence during <i>in vitro</i> maturation of mouse follicles

Kim, Yong Jin,Ku, Seung-Yup,Kim, Yoon Young,Liu, Hung Ching,Chi, Sung Wook,Kim, Seok Hyun,Choi, Young Min,Kim, Jung Gu,Moon, Shin Yong Oxford University Press 2013 Human reproduction Vol.28 No.11

<P><B>STUDY QUESTION</B></P><P>Do microRNAs (miRNAs) in granulosa cells (GCs) affect oocyte maturation during ovarian follicle development?</P><P><B>SUMMARY ANSWER</B></P><P>Sophisticated regulation by miRNAs in ovarian GCs may improve oocyte maturation efficiency during ovarian follicle development.</P><P><B>WHAT IS KNOWN ALREADY</B></P><P>The meiotic competence of oocytes depends on the follicle's potential to undergo appropriate maturation and is an important factor in infertility therapies such as IVF. The exact function of the GCs during follicular development remains unknown.</P><P><B>STUDY DESIGN, SIZE, DURATION</B></P><P>After <I>in vitro</I> maturation (IVM) and ovulation induction of isolated ovarian pre-antral follicles from 12-day-old female C57BL6 mice (<I>n</I> = 40), miRNA expression in the GCs was compared according to the maturity of the oocyte (metaphase I (MI) versus metaphase II (MII)). The miRNAs, which showed notable different expression, were modulated by transfection during IVM of follicles.</P><P><B>MATERIALS, SETTING, METHODS</B></P><P>miRNA expression and candidate target gene expression in GCs of isolated murine ovarian pre-antral follicles were evaluated by real-time PCR after IVM. miR mimics and -inhibitors for selected miRNAs were transfected into the <I>in vitro</I>-maturated follicles, and ovulation, oocyte maturation and fertilization rates were compared. Candidate target gene expressions in GC were evaluated by quantitative PCR and immunohistochemistry using confocal microscopy.</P><P><B>MAIN RESULTS AND THE ROLE OF CHANCE</B></P><P>The relative expression of mmu-let-7b (0.78 ± 0.10, <I>P</I> = 0.016), mmu-let-7c (0.78 ± 0.12, <I>P</I> = 0.029), mmu-miR-27a (0.57 ± 0.18, <I>P</I> = 0.016) and mmu-miR-322 (0.59 ± 0.14, <I>P</I> = 0.008) was significantly lower in the GCs of follicles containing MII oocytes compared with those of MI oocytes. Transfection with a mmu-miR-27a-mimic sequence decreased the oocyte maturation rate compared with that for the control (9.4 versus 18.9%, <I>P</I> = 0.042), and transfection with mmu-let-7c-, mmu-miR-27a- and mmu-miR-322-inhibitor sequences increased the oocyte maturation rate by 1.5- to 2.0-folds compared with that for the control (40.6, 31.6, and 30.5%versus 18.9%, <I>P</I> < 0.001, <I>P</I> = 0.013, <I>P</I> = 0.021, respectively). The expression of IGFBP-2 was higher in GCs of MII than in the GCs of MI, and higher in miR-inhibitor transfection groups than in miR-mimic transfection groups and controls.</P><P><B>LIMITATIONS, REASONS FOR CAUTION</B></P><P>An <I>in vitro</I> model was used in lieu of an <I>in vivo</I> model because of the ease of performing miRNA transfection in cell culture. However, studies have shown similarities and differences in <I>in vivo</I> versus <I>in vitro</I> cultured follicles. The findings of the present study need to be confirmed using <I>in vivo</I> maturation models and extended to evaluate developmental competence.</P><P><B>WIDER IMPLICATIONS OF THE FINDINGS</B></P><P>Our findings suggest that sophisticated miRNA regulation in GCs may improve oocyte maturation efficiency during ovarian follicle development.</P><P><B>STUDY FUNDING/COMPETING INTEREST(S)</B></P><P>This work was supported by a grant from the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A111539). None of the authors has any conflicts of interest to declare.</P>

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.

Endoplasmic Stress Inhibition during Oocyte Maturation Improves Preimplantation Development of Cloned Pig Embryos

ELAHI MD FAZLE,이은송,신혜지,이주형 사단법인 한국동물생명공학회 2017 한국동물생명공학회지 Vol.32 No.4

Mitochondrial dysfunction is found in oocytes and transmitted to offspring due to maternal obesity. Treatment of obese mothers with endoplasmic reticulum (ER) stress inhibitors such as salubrinal (SAL) can reverse the mitochondrial dysfunction and result in normal embryonic development. Pig oocytes have also shown ER stress mostly in metaphase II stage. ER stress in oocytes may hinder the in vitro production of pig embryos. This study investigated the effect of ER stress inhibition by SAL treatment during in vitro maturation (IVM) of porcine oocytes at 1, 10, 50 and 100 nM concentrations. Firstly, we tested various concentrations of SAL. SAL at 10 nM showed higher (P< 0.05) developmental competence to the blastocyst stage (55.6%) after parthenogenesis (PA) than control (44.2%) while not different from other concentrations (49.2, 51.6, and 50.8% for 1, 50, and 100 nM, respectively). Secondly, we performed time-dependent treatment at 10 nM of SAL for IVM of oocytes. It revealed that treatment with SAL during 22 to 44 h of IVM significantly improved PA embryonic development to the blastocyst stage compared to control (40.5, 46.3, 51.7 and 60.2% for control, 0 to 22 h, 22 to 44 h and 0 to 44 h of IVM, respectively, P < 0.05). Glutathione (GSH) content is an indicator of cytoplasmic maturation of oocytes. Reactive oxygen species (ROS) have a harmful effect on developmental competence of oocytes. For this, we determined the intraoocyte levels of GSH and ROS after 44 h of IVM. It was found that SAL increased intraoocyte GSH level and also decreased ROS level (P < 0.05). Finally, we performed somatic cell nuclear transfer (SCNT) after treating oocytes with 10 nM SAL during IVM. SAL treatment significantly improved blastocyst formation of SCNT embryos compared to control (39.6% vs. 24.7%, P < 0.05). Our results indicate that treatment of pig oocytes with ER stress inhibitor SAL during IVM improves preimplantation development PA and cloned pig embryos by influencing cytoplasmic maturation in terms of increased GSH content and decreased ROS level in IVM pig oocytes.

Stage-specific Expression of Lanosterol 14${\alpha}$-Demethylase in Mouse Oocytes in Relation to Fertilization and Embryo Development Competence

Song, Xiaoming,Ouyang, Hong,Tai, Ping,Chen, Xiufen,Xu, Baoshan,Yan, Jun,Xia, Guoliang,Zhang, Meijia Asian Australasian Association of Animal Productio 2009 Animal Bioscience Vol.22 No.3

Follicular fluid meiosis-activating sterol (FF-MAS) has been suggested as a positive factor which could improve the oocyte quality and subsequent embryo development after in vitro fertilization. However, FF-MAS is a highly lipophilic substance and is hard to detect in studying the relationship between MAS and quality of oocyte maturation. The present study focused on the expression of lanosterol 14${\alpha}$-demethylase (LDM), a key enzyme that converts lanosterol to FF-MAS, on mouse oocyte maturation and its potency on development. LDM expression was strong in gonadotropin-primed germinal vesicle stage oocytes, weak after germinal vesicle breakdown (GVBD), and then strong in MII stage oocytes. The LDM-specific inhibitor azalanstat significantly inhibited oocyte fertilization (from 79.4% to 68.3%, p<0.05). Also, azalanstat (5 to 50 ${\mu}M$) decreased the percentage of blastocyst development dosedependently (from 78.7% to 23.4%, p<0.05). The specific inhibition of sterol ${\Delta}14$-reductase and ${\Delta}7$-reductase by AY9944 accumulates FF-MAS and could increase blastocyst development rates. Additionally, in the AY9944 group, the rate of inner cell mass (ICM)/ total cells was similar to that of in vivo development, but the rate was significantly decreased in azalanstat treatment. In conclusion, LDM, the key enzyme of FF-MAS production, may play an important role in fertilization and early development of the mouse embryo, especially in vitro.

OPU 채란계절이 한우의 난자 품질 및 발달 능력에 미치는 영향

김성수,최병현,조현태,진종인,하아나,민찬식,조규완,공일근 사단법인 한국동물생명공학회 2014 한국동물생명공학회지 Vol.29 No.3

Implementation of smart embryo technologies in cattle e.g. ovum pick-up followed by in vitro embryo production(OPU-IVP). Seasonal variation is important factor for follicular growth, oocytes quality, quantity and developmentalcompetence. Therefore the aim of present study was carried out to investigated whether the seasons (hot and cool) effecton follicular development, oocyte recovery and subsequent embryo development. Follicular oocytes were aspirated fromKorean native cows (Hanwoo) by the ovum pick-up (OPU) method, which was performed 24 times during two differentseasons, the hot (July to September) and cool (October to December), from OPU donors. The recovered oocytes wereclassified according to morphological categories and used for in vitro embryo production (IVEP). The mean numberof total follicles was significantly higher (p<0.05) during the hot season (18.32 ± 2.26) compared to cool season (15.41± 3.34). Furthermore, seasons did not significantly effect on the number of oocytes recovered (hot season: 41.16%vs. cool season: 46.14%). However, the average number of Grade A oocytes was significantly greater during hot (1.75± 1.86) season compared to the cool season (1.00 ± 1.46), but there was no significant difference of other gradesoocytes. The cleavage rate (hot: 66.67% vs. cool: 63.3%) and embryo development (hot: 58.95% vs. cool: 56.97%)did not differ significantly between the seasons. In conclusion, the results of present study suggest that the season(hot and cool) does not have effects on the oocyte recovery and embryo developmental competence of in vitro culturedembryos.

In vitro maturation using αMEM with reduced NaCl enhances maturation and developmental competence of pig oocytes after somatic cell nuclear transfer

Yongjin Lee,Joohyeong Lee,Sang-Hwan Hyun,Geun-Shik Lee,Eunsong Lee 대한수의학회 2022 Journal of Veterinary Science Vol.23 No.2

Background: Compared to medium containing 108 mM sodium chloride (NaCl), in vitro maturation (IVM) using a simple medium with reduced (61.6 mM) NaCl increases the cytoplasmic maturation and embryonic development of pig oocytes. Objectives: This study determines the effect of a complex medium containing reduced NaCl on the IVM and embryonic development of pig oocytes. Methods: Pig oocytes were matured in Minimum Essential Medium Eagle-alpha modification (αMEM) supplemented with 61.6 (61αMEM) or 108 (108αMEM) mM NaCl, and containing polyvinyl alcohol (PVA) (αMEMP) or pig follicular fluid (PFF) (αMEMF). Medium-199 (M199) served as the control for conventional IVM. Cumulus cell expansion, nuclear maturation, intra-oocyte glutathione (GSH) contents, size of perivitelline space (PVS), and embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) were evaluated after IVM. Results: Regardless of PVA or PFF supplementation, oocytes matured in 61αMEM showed increased intra-oocyte GSH contents and width of PVS (p < 0.05), as well as increased blastocyst formation (p < 0.05) after PA and SCNT, as compared to oocytes matured in 108αMEMP and M199. Under conditions of PFF-enriched αMEM, SCNT oocytes matured in 61αMEMF showed higher blastocyst formation (p < 0.05), compared to maturation in 108αMEMF and M199, whereas PA cultured oocytes showed no significant difference. Conclusions: IVM in αMEM supplemented with reduced NaCl (61.6 mM) enhances the embryonic developmental competence subsequent to PA and SCNT, which attributes toward improved oocyte maturation.

탈핵 후 동결한 MII 난자의 활성화 시기가 체세포 핵치환 이후 소 난자의 체외발달에 미치는 영향

박세필,김은영,김선균,이영재,길광수,박세영,윤지연,이창현,정길생 한국동물번식학회 2002 Reproductive & developmental biology Vol.26 No.3

This study was to evaluate the in vitro survival of bovine enucleated MII (eMII) oocytes according to minimum volume cooling (MVC) freezing method and activation timing, and their in vitro development after somatic cell nuclear transfer (SONT). in vitro matured bovine oocytes for 20 h were stained with 5 $\mu\textrm{g}$/$m\ell$ Hoechst, and their 1st polar body and MII plate were removed by enucleation micropipette under UV filter. Also, eMII oocytes were subjected to activation after (group II) and before (group III) vitrification in 5 ${\mu}{\textrm}{m}$ ionomycin added CRlaa medium for 5 min. For vitrification, eMll oocytes were pretreated with EG10 for 5 min, exposed to EG30 for 30 sec and then directly plunged into L$N_2$. Thawing was taken by 4-step procedures at 37$^{\circ}C$. Survived eMII oocytes were subjected to SONT with cultured adult bovine ear cells. Reconstructed oocytes were cultured in 10 $\mu\textrm{g}$/$m\ell$ of cycloheximide and 2.5 $\mu\textrm{g}$/$m\ell$ of cytochalasin D added CRlaa medium for 1 h, and then in 10 $\mu\textrm{g}$/$m\ell$ of cycloheximide added CRlaa medium for 4 h. Subsequently, the reconstructed oocytes were incubated for 2 days and cleaved embryos were further cultured on cumulus-cell monolayer drop in CRlaa medium for 6 days. Survival rates of bovine vitrified-thawed eMII oocytes in group II (activation after vitrification and thawing) and III (activation before vitrification) were 81.0% and 84.9%, respectively. Fusion rates of cytoplasts and oocytes in group II and III were 69.0% and 70.0%, respectively, and their results were not different with non-frozen NT group (control, 75.2%). Although their cleaved rates (53.4% and 58.4%) were not different, cytoplasmic fragment rate in group II (32.8%) was significantly higher than that in group III (15.6%)(P<0.05). Also, subsequent development rate into >morula in group II (8.6%) was low than that in group III(15.6%). However, in vitro development rate in group III was not different with that in control (24.8%). This result suggested that MVC method was appropriate freezing method for the bovine eMII oocytes and vitrified eMII oocytes after pre-activation could support in vitro embryonic development after SONT as equally well as fresh oocytes.

Briliant Cresyl Blue 염색 방법과 극체 방출 여부에 따른 돼지 체외수정용 난포란 선별 방법이 배발달에 미치는 영향

김연수,김철욱,김인철,곽대오,정기화 한국동물생명공학회(구 한국동물번식학회) 2009 Reproductive & developmental biology Vol.33 No.1

The brilliant cresyl blue (BCB) has been used to select the developmental competent oocytes in pigs, goats and cows. Growing oocytes have a higher level of active glucose-6-phosphate dehydrogenase(G6PDH) compare to mature oocytes and are rarely stained compared to mature oocytes, because G6PDH converts BCB to colorless. First polar body extrusion regard as a guideline of meoisis completion. Selection of polar body extrude oocyte is more developmental competent to blastocyst than unselected. This study was conducted to compare the BCB test to the polar body extrusion on selection of developmental competent porcine oocytes for the production of blastocyst. Cumulus-Oocytes complex were exposed to 26uM BCB stain diluted in NCSU-23 for 90 min. There was no significant difference embryo development to blastocysts between BCB treated and not treated(19.58±1.99 vs 18.75±2.27 %), which means there was no detrimental effect of BCB exposure to oocytes. Normal fertilization is not differed among treatment groups from 70.0 to 78.4% development to blastocyst, beside polyspermy did not. To compare two different selection methods, BCB test and polar body extrusion, evaluate the developmental competent of IVP embryos. BCB+PB+(blue stained and polar body extruded, 20.71±0.45%) and BCB-PB+(colorless and polar body extruded, 20.04±1.29%) groups are significantly (p<0.05) higher developed than those of BCB+PB-(blue stained and no polar body, 13.24±0.73%) and BCB-PB-(colorless and no poladbody, 7.25±0.77%). These results showed that selection of polar body extruded oocytes method is more efficient than that of BCB test.