Melatonin promotes the development of sheep transgenic cloned embryos by protecting donor and recipient cells

Yao, Yujun; Yang, Ailing; Wu, Hao; Deng, Shoulong; Yang, Hai; Ma, Wenkui; Lv, Dongying; Fu, Yao; Ji, Pengyun; Tan, Xinxing; Zhao, Wei; Lian, Zhengxing; Zhang, Lu; Liu, Guoshi

doi:10.1080/15384101.2022.2051122

Cited by 6 publications

(4 citation statements)

References 58 publications

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“…Overall, unraveling and comparing the intra-and extra-embryonic factors that affect the molecular mechanisms underlying cell commitment and the inter-proteomic crosstalk between transcription factors that dictate cell fate, such as CDX-2-, SOX-2-, and GATA-6, seems to be an important aspect of promoting improvement in the efficiency of generating ex vivo-derived high-quality blastocysts in equids and other mammalian species. This might also enhance in vivo developmental competence after intrauterine transfer of IVP-derived embryos propagated by advanced assisted reproductive technologies (ARTs), such as in vitro fertilization (IVF), based either on gamete co-incubation or intracytoplasmic sperm injection (ICSI) [44][45][46][47], and cloning based on somatic cell nuclear transfer (SCNT) [48][49][50].…”

Section: Discussionmentioning

confidence: 99%

In Vitro-Produced Equine Blastocysts Exhibit Greater Dispersal and Intermingling of Inner Cell Mass Cells than In Vivo Embryos

Umair

Scheeren

Beitsma

et al. 2023

IJMS

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In vitro production (IVP) of equine embryos is increasingly popular in clinical practice but suffers from higher incidences of early embryonic loss and monozygotic twin development than transfer of in vivo derived (IVD) embryos. Early embryo development is classically characterized by two cell fate decisions: (1) first, trophectoderm (TE) cells differentiate from inner cell mass (ICM); (2) second, the ICM segregates into epiblast (EPI) and primitive endoderm (PE). This study examined the influence of embryo type (IVD versus IVP), developmental stage or speed, and culture environment (in vitro versus in vivo) on the expression of the cell lineage markers, CDX-2 (TE), SOX-2 (EPI) and GATA-6 (PE). The numbers and distribution of cells expressing the three lineage markers were evaluated in day 7 IVD early blastocysts (n = 3) and blastocysts (n = 3), and in IVP embryos first identified as blastocysts after 7 (fast development, n = 5) or 9 (slow development, n = 9) days. Furthermore, day 7 IVP blastocysts were examined after additional culture for 2 days either in vitro (n = 5) or in vivo (after transfer into recipient mares, n = 3). In IVD early blastocysts, SOX-2 positive cells were encircled by GATA-6 positive cells in the ICM, with SOX-2 co-expression in some presumed PE cells. In IVD blastocysts, SOX-2 expression was exclusive to the compacted presumptive EPI, while GATA-6 and CDX-2 expression were consistent with PE and TE specification, respectively. In IVP blastocysts, SOX-2 and GATA-6 positive cells were intermingled and relatively dispersed, and co-expression of SOX-2 or GATA-6 was evident in some CDX-2 positive TE cells. IVP blastocysts had lower TE and total cell numbers than IVD blastocysts and displayed larger mean inter-EPI cell distances; these features were more pronounced in slower-developing IVP blastocysts. Transferring IVP blastocysts into recipient mares led to the compaction of SOX-2 positive cells into a presumptive EPI, whereas extended in vitro culture did not. In conclusion, IVP equine embryos have a poorly compacted ICM with intermingled EPI and PE cells; features accentuated in slowly developing embryos but remedied by transfer to a recipient mare.

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Section: Discussionmentioning

confidence: 99%

In Vitro-Produced Equine Blastocysts Exhibit Greater Dispersal and Intermingling of Inner Cell Mass Cells than In Vivo Embryos

Umair

Scheeren

Beitsma

et al. 2023

IJMS

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“…Summing up, the present study has provided strong scientific evidence and mechanistic insights into the pivotal role of melatonin as an extrinsic agent that not only decreases the intracellular levels of oxygen free radicals but also impedes/relieves the processes of oxidative stress in the in vitro cultured IVF-derived pig embryos. Considering the abovementioned findings, the advantageous effect of exogenous melatonin ought to be more widely adopted among laboratories working with IVP porcine (and other species) embryos, including those IVP embryos generated using intracytoplasmic sperm injection (ICSI) [ 98 , 99 , 100 ] and cloning by SCNT into enucleated oocytes [ 101 , 102 , 103 ].…”

Section: Discussionmentioning

confidence: 99%

Exogenous Melatonin in the Culture Medium Does Not Affect the Development of In Vivo-Derived Pig Embryos but Substantially Improves the Quality of In Vitro-Produced Embryos

et al. 2022

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Cloned and transgenic pigs are relevant human disease models and serve as potential donors for regenerative medicine and xenotransplantation. These technologies demand oocytes and embryos of good quality. However, the current protocols for in vitro production (IVP) of pig embryos give reduced blastocyst efficiency and embryo quality compared to in vivo controls. This is likely due to culture conditions jeopardizing embryonic homeostasis including the effect of reactive oxygen species (ROS) influence. In this study, the antioxidant melatonin (1 nM) in the maturation medium, fertilization medium, or both media was ineffective in enhancing fertilization or embryonic development parameters of in vitro fertilized oocytes. Supplementation of melatonin in the fertilization medium also had no effect on sperm function. In contrast, the addition of melatonin to the embryo culture medium accelerated the timing of embryonic development and increased the percentages of cleaved embryos and presumed zygotes that developed to the blastocyst stage. Furthermore, it increased the number of inner mass cells and the inner mass cell/total cell number ratio per blastocyst while increasing intracellular glutathione and reducing ROS and DNA damage levels in embryos. Contrarily, the addition of melatonin to the embryo culture medium had no evident effect on in vivo-derived embryos, including the developmental capacity and the quality of in vivo-derived 4-cell embryos or the percentage of genome-edited in vivo-derived zygotes achieving the blastocyst stage. In conclusion, exogenous melatonin in the embryo culture medium enhances the development and quality of in vitro-derived embryos but not in in vivo-derived embryos. Exogenous melatonin is thus recommended during embryo culture of oocytes matured and fertilized in vitro for improving porcine IVP efficiency.

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“…Activated iSCNT-derived embryos were cultured in NCSU-23 [ 30 ], PZM-3 [ 11 ], or PZM-5 [ 23 ] droplets covered with mineral oil (10–15 embryos/droplets). These were cultured for 7 days in an incubator with an environment of 5% CO₂, 5% O 2 , and 90% N 2 at a temperature of 39°C.…”

Section: Methodsmentioning

confidence: 99%

“…In this context, an important role is also played by the strategies used to artificially synchronize the mitotic cycle of ex vivo-expanded nuclear donor cells at the G0/G1 stages [ 8 – 10 ]. It is also noteworthy that the developmental outcome of somatic cell-cloned embryos is largely determined by the molecular quality parameters reflected in the incidence of apoptotic cell death and oxidative stress processes in the in vitro cultured nuclear donor cells and SCNT-derived embryos [ 11 , 12 ]. Moreover, the developmental capability of cloned embryos is remarkably affected by the molecular quality of metaphase II-stage nuclear recipient oocytes, which depends largely on coordination between the processes of meiotic, cytoplasmic, and epigenomic maturation [ 13 – 15 ].…”

Section: Introductionmentioning

confidence: 99%

iSCNT embryo culture system for restoration of Cervus nippon hortulorum, presumed to be sika deer in the Korean Peninsula

Park,

Oh,

Kim

2024

PLoS ONE

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Sika deer inhabiting South Korea became extinct when the last individual was captured on Jeju Island in Korea in 1920 owing to the Japanese seawater relief business, but it is believed that the same subspecies (Cervus nippon hortulorum) inhabits North Korea and the Russian Primorskaya state. In our study, mt-DNA was used to analyze the genetic resources of sika deer in the vicinity of the Korean Peninsula to restore the extinct species of continental deer on the Korean Peninsula. In addition, iSCNT was performed using cells to analyze the potential for restoration of extinct species. The somatic cells of sika deer came from tissues of individuals presumed to be Korean Peninsula sika deer inhabiting the neighboring areas of the Primorskaya state and North Korea. After sequencing 5 deer samples through mt-DNA isolation and PCR, BLAST analysis showed high matching rates for Cervus nippon hortulorum. This shows that the sika deer found near the Russian Primorsky Territory, inhabiting the region adjacent to the Korean Peninsula, can be classified as a subspecies of Cervus nippon hortulorum. The method for producing cloned embryos for species restoration confirmed that iSCNT-embryos developed smoothly when using porcine oocytes. In addition, the stimulation of endometrial cells and progesterone in the IVC system expanded the blastocyst cavity and enabled stable development of energy metabolism and morphological changes in the blastocyst. Our results confirmed that the individual presumed to be a continental deer in the Korean Peninsula had the same genotype as Cervus nippon hortulorum, and securing the individual’s cell-line could restore the species through replication and produce a stable iSCNT embryo.

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Melatonin promotes the development of sheep transgenic cloned embryos by protecting donor and recipient cells

Cited by 6 publications

References 58 publications

In Vitro-Produced Equine Blastocysts Exhibit Greater Dispersal and Intermingling of Inner Cell Mass Cells than In Vivo Embryos

In Vitro-Produced Equine Blastocysts Exhibit Greater Dispersal and Intermingling of Inner Cell Mass Cells than In Vivo Embryos

Exogenous Melatonin in the Culture Medium Does Not Affect the Development of In Vivo-Derived Pig Embryos but Substantially Improves the Quality of In Vitro-Produced Embryos

iSCNT embryo culture system for restoration of Cervus nippon hortulorum, presumed to be sika deer in the Korean Peninsula

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