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Molecular Mechanisms of Somatic Cell Cloning and Other Assisted Reproductive Technologies in Mammals

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Biology".

Deadline for manuscript submissions: closed (20 November 2024) | Viewed by 10176

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Prof. Dr. Marcin Samiec

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Guest Editor

Department of Reproductive Biotechnology and Cryoconservation, National Research Institute of Animal Production, Balice near Kraków, 32-083 Balice, Poland
Interests: reproductive biology and biotechnology in different mammalian species (especially pigs, goats, cattle, rabbits and horses); assisted reproductive technologies (ARTs)—experimental and applied embryology; intra- and interspecies somatic cell cloning by somatic cell nuclear transfer (SCNT); transgenic research; parthenogenetic activation of oocytes (methods and molecular mechanisms of oocyte activation); in vitro embryo production (IVP)—in vitro oocyte maturation (IVM), in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), in vitro embryo culture (IVC); stem cell research; epigenetic and molecular aspects of embryonic development—epigenetics in developmental biology, epigenetic modulation of nuclear donor cells (somatic cells, stem cells), SCNT-derived oocytes or cloned embryos; unravelling the transcriptomic and proteomic profiles in nuclear donor cell lines (somatic and stem cell lines) and nuclear recipient oocytes; the ART-mediated programs focused on biotechnological, transgenic and biomedical research
Special Issues, Collections and Topics in MDPI journals

Prof. Dr. Maria Skrzyszowska

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Guest Editor

Department of Reproductive Biotechnology and Cryoconservation, National Research Institute of Animal Production, Balice near Kraków, 32-083 Balice, Poland
Interests: animal reproduction biotechnology; somatic cell cloning in different mammalian species; embryonic cell cloning in different mammalian species, including embryo bisection methods; in vitro embryo production; in vitro oocyte maturation and microsurgical in vitro fertilization by ICSI; transgenesis; modern strategies to assess the molecular quality of somatic cell lines and female gametes for the purposes of advanced ARTs
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Although approximately 25 mammalian species have been cloned by somatic cell nuclear transfer (SCNT), the efficiency of somatic cell cloning is still at a disappointingly low level, ranging from 0.1% to less than 5% as measured by the percentage of the cloned progeny generated to the number of oocytes reconstructed by SCNT. In contrast to SCNT-based cloning, other assisted reproductive technologies (ARTs) encompassing ex vivo embryo production with the use of either conventional in vitro fertilization (IVF) by gamete co-incubation or microsurgical IVF by intracytoplasmic sperm injection (ICSI) are most frequently characterized not only by higher rates of embryo- and fetogenesis, but also by higher pregnancy and offspring outcomes in different mammalian species. Nonetheless, the parameters of embryonic and fetal development and perinatal survival that are pinpointed for SCNT-, IVF-, and ICSI-mediated in vitro embryo production (IVP) tend to dwindle considerably as compared to the ARTs based on the methods of in vivo embryo production. For these reasons, pivotal efforts aimed both to improve the pre- and/or post-implantation capabilities and to enhance the molecular quality of mammalian cloned and other in vitro-produced embryos need to be undertaken and seem to play a very preponderant role in a wide variety of ARTs. This finding appears to be a sine qua non condition not only for augmenting the efficacies noticed for SCNT-based cloning and other ARTs, but also for accelerating and intensifying broad-spectrum application of these strategies to embryological, biotechnological, transgenic and biomedical research.

In summary, this Special Issue provides the possibility to publish research articles, comprehensive reviews and short communications focused on exploring or recognizing an extensive range of biological, molecular, genetic and epigenetic determinants profoundly affecting the efficiencies of somatic cell cloning and other ARTs at the levels of nuclear donor cells (somatic and stem cells), male and female gametes (spermatozoa and oocytes) and in vitro- or in vivo-produced embryos. Thorough identification of the aforementioned determinants can contribute greatly to the amelioration, optimization and implementation of SCNT-, IVF-, and ICSI-mediated IVP approaches as well as a wide array of ARTs involving the procedures of in vivo embryo production. This is the predominant prerequisite for the enhancement of practically applying the above-indicated strategies as important, reliable and feasible ARTs for the purposes of experimental and applied embryology, biotechnology, ex situ biodiversity preservation, transgenics, biomedicine, biopharmacy and the creation of animal models for the etiopathogenesis and pathophysiology of human diseases.

Prof. Dr. Marcin Samiec
Prof. Dr. Maria Skrzyszowska
Guest Editors

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Keywords

somatic cell nuclear transfer (SCNT)
SCNT-based cloning
assisted reproductive technology (ART)
in vitro embryo production (IVP)
in vitro fertilization (IVF)
intracytoplasmic sperm injection (ICSI)
in vivo embryo production

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Published Papers (5 papers)

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Research

21 pages, 3311 KiB

Open AccessArticle

Anabolic Steroids Activate the NF-κB Pathway in Porcine Ovarian Putative Stem Cells Independently of the ZIP-9 Receptor

by Kamil Wartalski, Jerzy Wiater, Patrycja Maciak, Agnieszka Pastuła, Grzegorz J. Lis, Marcin Samiec, Monika Trzcińska and Małgorzata Duda

Int. J. Mol. Sci. 2024, 25(5), 2833; https://doi.org/10.3390/ijms25052833 - 29 Feb 2024

Cited by 4 | Viewed by 1351

Abstract

Boldenone (Bdn) and nandrolone (Ndn) are anabolic androgenic steroids (AASs) that, as our previous studies have shown, may increase the risk of neoplastic transformation of porcine ovarian putative stem cells (poPSCs). The NF-κB pathway may be important in the processes of carcinogenesis and tumour progression. Therefore, in this work, we decided to test the hypothesis of whether Bdn and Ndn can activate the NF-κB pathway by acting through the membrane androgen receptor ZIP-9. For this purpose, the expression profiles of both genes involved in the NF-κB pathway and the gene coding for the ZIP-9 receptor were checked. The expression and localization of proteins of this pathway in poPSCs were also examined. Additionally, the expression of the ZIP-9 receptor and the concentration of the NF-κB1 and 2 protein complex were determined. Activation of the NF-κB pathway was primarily confirmed by an increase in the relative abundances of phosphorylated forms of RelA protein and IκBα inhibitor. Reduced quantitative profiles pinpointed not only for genes representing this pathway but also for unphosphorylated proteins, and, simultaneously, decreased concentration of the NF-κB1 and 2 complex may indicate post-activation silencing by negative feedback. However, the remarkably and sustainably diminished expression levels noticed for the SLC39A9 gene and ZIP-9 protein suggest that this receptor does not play an important role in the regulation of the NF-κB pathway. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Somatic Cell Cloning and Other Assisted Reproductive Technologies in Mammals)

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15 pages, 4763 KiB

Open AccessArticle

Early Pregnancy Markers in the Serum of Ewes Identified via Proteomic and Metabolomic Analyses

by Yaying Zhai, Fan Xia, Luting Shi, Wenkui Ma, Xiaoyang Lv, Wei Sun, Pengyun Ji, Shuai Gao, Zoltan Machaty, Guoshi Liu and Lu Zhang

Int. J. Mol. Sci. 2023, 24(18), 14054; https://doi.org/10.3390/ijms241814054 - 13 Sep 2023

Cited by 2 | Viewed by 1792

Abstract

The diagnosis of ewes’ pregnancy status at an early stage is an efficient way to enhance the reproductive output of sheep and allow producers to optimize production and management. The techniques of proteomics and metabolomics have been widely used to detect regulatory factors in various physiological processes of animals. The aim of this study is to explore the differential metabolites and proteins in the serum of pregnant and non-pregnant ewes by proteomics and metabolomics. The serum of ewes at 21, 28 and 33 days after artificial insemination (AI) were collected. The pregnancy stratus of the ewes was finally determined through ultrasound examination and then the ewes were grouped as Pregnant (n = 21) or N on-pregnant (n = 9). First, the serum samples from pregnant or non-pregnant ewes at 21 days after AI were selected for metabolomic analysis. It was found that the level of nine metabolites were upregulated and 20 metabolites were downregulated in the pregnant animals (p < 0.05). None of these differential metabolomes are suitable as markers of pregnancy due to their small foldchange. Next, the proteomes of serum from pregnant or non-pregnant ewes were evaluated. At 21 days after AI, the presence of 321 proteins were detected, and we found that the level of three proteins were upregulated and 11 proteins were downregulated in the serum of pregnant ewes (p < 0.05). The levels of serum amyloid A (SAA), afamin (AFM), serpin family A member 6 (SERPINA6) and immunoglobulin-like domain-containing protein between pregnant and non-pregnant ewes at 21-, 28- and 33-days post-AI were also analyzed via enzyme-linked immunosorbent assay (ELISA). The levels of SAA and AFM were significantly higher in pregnant ewes than in non-pregnant ewes, and could be used as markers for early pregnancy detection. Overall, our results show that SAA and AFM are potential biomarkers to determine the early pregnancy status of ewes. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Somatic Cell Cloning and Other Assisted Reproductive Technologies in Mammals)

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16 pages, 18708 KiB

Open AccessArticle

Porcine Follicular Fluid-Derived Exosome: The Pivotal Material for Porcine Oocyte Maturation in Lipid Antioxidant Activity

by Euihyun Kim, Kihae Ra, Myung-Shin Lee and Geon A. Kim

Int. J. Mol. Sci. 2023, 24(12), 9807; https://doi.org/10.3390/ijms24129807 - 6 Jun 2023

Cited by 3 | Viewed by 2265

Abstract

Several studies have examined exosomes derived from porcine follicular fluid (FF), but few have reported their application in controlled experiments. The main concern in the field of embryology may be that controlled conditions, such as using a defined medium intermittently, cause poor results in mammalian oocyte maturation and embryo development. The first reason is the absence of the FF, which copes with the majority of the processes emerging in oocytes and embryos. Therefore, we added exosomes derived from porcine FF to the maturation medium of porcine oocytes. For morphological assessment, cumulus cell expansion and subsequent embryonic development were evaluated. Moreover, several stainings, such as glutathione (GSH) and reactive oxygen species (ROS), fatty acid, ATP, and mitochondrial activity, as well as evaluations of gene expression and protein analysis, were used for the functional verification of exosomes. When the oocytes were treated with exosomes, the lipid metabolism and cell survival of the oocytes were fully recovered, as well as morphological evaluations compared to the porcine FF-excluded defined medium. Therefore, controlled experiments may produce reliable data if the exosomes are treated with the desired amounts, and we suggest applying FF-derived exosomes to promote experimental data when performing controlled experiments in embryology. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Somatic Cell Cloning and Other Assisted Reproductive Technologies in Mammals)

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17 pages, 3680 KiB

Open AccessArticle

Identification of Important Factors Causing Developmental Arrest in Cloned Pig Embryos by Embryo Biopsy Combined with Microproteomics

by Yuxing Zhang, Liusong Yang, Yiqian Zhang, Yalin Liang, Huaxing Zhao, Yanan Li, Gengyuan Cai, Zhenfang Wu and Zicong Li

Int. J. Mol. Sci. 2022, 23(24), 15975; https://doi.org/10.3390/ijms232415975 - 15 Dec 2022

Cited by 1 | Viewed by 1783

Abstract

The technique of pig cloning holds great promise for the livestock industry, life science, and biomedicine. However, the prenatal death rate of cloned pig embryos is extremely high, resulting in a very low cloning efficiency. This limits the development and application of pig cloning. In this study, we utilized embryo biopsy combined with microproteomics to identify potential factors causing the developmental arrest in cloned pig embryos. We verified the roles of two potential regulators, PDCD6 and PLK1, in cloned pig embryo development. We found that siRNA-mediated knockdown of PDCD6 reduced mRNA and protein expression levels of the pro-apoptotic gene, CASP3, in cloned pig embryos. PDCD6 knockdown also increased the cleavage rate and blastocyst rate of cloned porcine embryos. Overexpression of PLK1 via mRNA microinjection also improved the cleavage rate of cloned pig embryos. This study provided a new strategy to identify key factors responsible for the developmental defects in cloned pig embryos. It also helped establish new methods to improve pig cloning efficiency, specifically by correcting the expression pattern of PDCD6 and PLK1 in cloned pig embryos. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Somatic Cell Cloning and Other Assisted Reproductive Technologies in Mammals)

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14 pages, 2009 KiB

Open AccessArticle

Knockdown of YY1 Inhibits XIST Expression and Enhances Cloned Pig Embryo Development

by Yazheng Dong, Xiao Wu, Xitong Peng, Liusong Yang, Baohua Tan, Huaxing Zhao, Enqin Zheng, Linjun Hong, Gengyuan Cai, Zhenfang Wu and Zicong Li

Int. J. Mol. Sci. 2022, 23(23), 14572; https://doi.org/10.3390/ijms232314572 - 23 Nov 2022

Cited by 2 | Viewed by 2060

Abstract

The technique of cloning has wide applications in animal husbandry and human biomedicine. However, the very low developmental efficiency of cloned embryos limits the application of cloning. Ectopic XIST-expression-induced abnormal X chromosome inactivation (XCI) is a primary cause of the low developmental competence of cloned mouse and pig embryos. Knockout or knockdown of XIST improves cloning efficiency in both pigs and mice. The transcription factor Yin yang 1(YY1) plays a critical role in XCI by triggering the transcription of X-inactive specific transcript (XIST) and facilitating the localization of XIST RNA on the X chromosome. This study aimed to investigate whether RNA interference to suppress the expression of YY1 can inhibit erroneous XIST expression, rescue abnormal XCI, and improve the developmental ability of cloned pig embryos. The results showed that YY1 binds to the 5′ regulatory region of the porcine XIST gene in pig cells. The microinjection of YY1 siRNA into cloned pig embryos reduced the transcript abundance of XIST and upregulated the mRNA level of X-linked genes at the 4-cell and blastocyst stages. The siRNA-mediated knockdown of YY1 altered the transcriptome and enhanced the in vitro and in vivo developmental efficiency of cloned porcine embryos. These results suggested that YY1 participates in regulating XIST expression and XCI in cloned pig embryos and that the suppression of YY1 expression can increase the developmental rate of cloned pig embryos. The present study established a new method for improving the efficiency of pig cloning. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Somatic Cell Cloning and Other Assisted Reproductive Technologies in Mammals)

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