Indian Journal of Animal Research

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Comparison of Gene Expression between Bovine Embryos Produced in vitro and Cloned Arabian Oryx Embryo from Bovine Ova

Ahmad R. Alhimaidi1, Maha H. Daghestani1, Muath Q. Al-Ghadi1, Ramzi A. Amran1, Afrah F. Alkhuriji1, Nawal M. Al-Malahi 1,*
1Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia.

ABSTRACT

Background: Embryonic biotechnology is an application used in animal production to expand the number of livestock animal species, provide animal products for global demand and preserve endangered species to improve reproduction, maintain biodiversity and genetic diversity and reduce the interval between generations. This animal production will supply goods and services for the human population’s well-being leading to economic growth. This biotechnology encompasses the use of assisted reproductive techniques (ARTs) like in vitro fertilization and nuclear transfer using somatic cells. This study compared the relative expression of some genes between the embryos that were fertilized in vitro (IVF) and cloned embryos by nuclear transfer of bovine (SCNT-B) and Arabian Oryx (SCNT-O).

Methods: The oocytes were matured, then fertilized and cultured for the IVF technique, while for the SCNT technique, the matured oocytes were enucleated to recipe the donor cells for bovine and Arabian Oryx then cultured, the embryos for both techniques were used for the gene expression of the studied genes.

Result: The expression of each studied gene (OCT4, DNMT1, BAX, GLUT1 and HSP70) was assessed in the early and late cleavage stages of the embryos produced by IVF in bovine, SCNT in bovine and SCNT in Arabian Oryx; this gene expression was compared between the three types of embryos. There was a non-significant difference in the relative expression between the embryos produced by the techniques mentioned above.

INTRODUCTION

Reproductive technology advancements like nuclear transfer, in vitro fertilization, artificial insemination and embryo transfer have significantly increased animal reproduction efficiency and are important tools for domestic and wild animal maintenance, especially for species that are endangered (Engdawork et al., 2024; Wolf et al., 2001).
 
In vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) or embryo cloning are two technologies with significant promise for producing a large number of domestic animal embryos for research purposes. Ova from slaughterhouses has supplied an essential source of embryos for several scientific objectives (Alofi and AlHimaidi, 2004).

In vitro fertilization technique from immature ovarian oocytes involves three essential stages maturation of the oocytes in vitro (IVM), in vitro fertilization (IVF) and in vitro culture of embryos (IVC) (Herrick et al., 2004;  Singh et al., 2020). The reprogramming for genes starts after fertilization by degrading oocyte-specific transcripts (maternal) that are not later expressed and replacing these maternal transcripts with embryonic transcripts, then promoting a significant reprogramming of the gene expression pattern in conjunction with the production of new transcripts that are not expressed in the oocyte (Schultz, 2002; Wrenzycki et al., 2005).

The nuclear transfer technique has several uses like breeding and clonal therapy. Although remarkable successes, the efficacy of SCNT remains low. Fibroblasts from various origins are frequently employed as donor cells for SCNT in domestic animals like sheep and cattle (100). SCNT provides an accurate genetic match by transferring the donor’s nucleus to the recipient oocyte enucleated (Keefer, 2015; Tian et al., 2003; Yun et al., 2008), after the nucleus has been transferred, the cloned embryo that results must grow to produce a genomic copy or clone of the individual whose nucleus was used, as well as reprogram the donor nucleus’ epigenetic markers to restore the proper gene expression patterns necessary for embryonic development to term. However, gene expression in cloned embryos is not always properly restored during reprogramming, resulting in incomplete donor nucleus reprogramming and abnormal expression of development-related genes (Park et al., 2003; Rodriguez-alvarez and Castro, 2010; EM et al., 2014). Remodeling and reprogramming are two distinct processes. Remodeling involves transforming the donor nucleus into one that resembles a zygote after being transported into the cytoplasm of a mature enucleated egg. Whereas reprogramming involves altering the chromatin structure (Whitworth and Prather, 2010). This nuclear transfer is dependent on the transplanted somatic cell nucleus becoming able to complete development from differentiated to totipotent stages (Matoba and Zhang, 2018).

During embryo development in mammals, two main reprogramming processes are known as epigenetic alteration. The first occurs during germ cell formation, while the second occurs during the preimplantation embryo development stages. Maternal to zygotic transition (MZT) is the designation for the second reprogramming process that occurs at the 8- to 16-cell stage in bovine which is important for the development of fertilized oocytes (Vigneault et al., 2004).

There are physiological changes that happen during the maternal-to-zygotic transition such as DNA methylation and chromatin structural remodeling and the new gene expression starts at MZT which means that the gene expression of the maternal turns off in the embryos produced by the IVF technique (Camargo et al., 2006), the expression of the somatic donor cell genes turns off in the embryos that produced by the SCNT technique throughout the transition from gametes into a pluripotent embryo and to the early differentiation of cell stages embryo  (Al-Malahi et al., 2022; EM et al., 2014).

The genes selected for this study OCT4 gene (Octamer-binding transcription factor-4), DNMT1gene (DNA methylation), BAX gene (apoptotic gene), GLUT1 gene (Glucose transporter) and HSP70 gene (cytoprotective factor) have a significant role in the development of good-quality embryos according to the reprogramming and remodeling (EM et al., 2014).

MATERIALS AND METHODS

Chemicals, reagents, media and tools for embryo production and gene analysis
 
All of them were from Sigma-Aldrich (St. Louis, MO, USA) unless defined otherwise.
 
Collection of ovaries and oocytes for maturation
 
The ovaries were collected from the bovine that was slaughtered in the slaughterhouse of Riyadh city to apply the in vitro fertilization and embryo cloning techniques, then the oocytes from these ovaries were collected and matured for 22-24 h in TCM-199 Earle’s salt maturation medium for 21-24 h at 39°C and 5% CO2 in the air with humidity as reported in (Al-Malahi et al., 2022; Ammari et al., 2022; Amran et al., 2024) for IVF and cloning techniques.
 
In vitro fertilization technique
 
The matured oocytes were fertilized in IVF-BO medium at 39°C and 5% CO2 for 24 h incubation in the air with humidity by the cryopreserved semen of the bull that was capacitated previously depending on (Al-Malahi et al., 2022; Amran et al., 2023; Canepa et al., 2014).
 
Nuclear transfer technique (cloning)
 
The matured oocytes were denuded by using hyaluronidase enzyme and the oocytes with the extruded first polar body were chosen for enucleation, the zona pellucida of the oocytes was dissected by pierced and then the 1st polar body was pushed out with the surrounding cytoplasm by using a glass needle under micromanipulator.  Adult Arabian oryx and bovine fibroblast somatic cells were used as donor cell sources after being cultured in (Dulbecco’s modified minimum essential medium DMEM) to the recipient enucleated oocyte, the nuclear fibroblast somatic cells were fused with the enucleated oocyte by two direct current pulses, a single AC pulse of 0.2 kV/cm for one second followed by a single DC pulse of 2.5 kV/cm for 50 μs in the cell fusion medium Zimmerman and then were activated for 5 min with 5 μM ionomycin at room temperature and followed by incubation in SOF medium for 6 hours at 38.5°C and 5% CO2, as detailed in (Al-Ghadi et al., 2020; Ammari, et al., 2022; Dinnyes et al., 2000).
 
Culture embryos

The fertilized and fused oocytes were cultured in IVC-SOF medium at 39°C in a 5% O2, 5% CO2 and 90% N2 for 7-9 days of incubation in the air with humidity (Alotaibi et al., 2022; Canepa et al., 2014).
 
Embryo collection and freezing
 
Embryos produced by both techniques mentioned were collected during the 7-9 days of culture in different cleavage stages from the early stages (2-4 and 8 cell stages) to the late stages (morula and blastocyst). These embryos were frozen in 0.1% PVA-PBS at -80°C until RNA extraction (Al-Malahi et al., 2022; Long et al., 2007).
 
Extraction of RNA and gene expression
 
The embryos’ RNA was extracted and gene expression for the genes HSP70 (heat shock protein), OCT4 (pluripotency gene), GLUT1 (glucose transporter), BAX (an apoptosis gene) and DNMT1 (DNA methyltransferase) were determined as detailed in our study (Al-Malahi et al., 2022). Table 1 displays the forward and reverse primers for the studied genes.

​

Table 1: Primers of the selected genes that were used to amplify by real-time polymerase chain reaction.



The comparative CT method was used to calculate the quantification of the expression levels gene concerning the GAPDH as a reference gene by the 2-ΔΔCt formula (Amarnath et al., 2007).

The experiment steps are viewed in (Fig 1). This study took 1 year and 5 months, it was done in the Zoology Department, Science College, KSU, Riyad, Saudi Arabia.

​

Fig 1: General view of the experiment steps that started from the collection of the immature oocytes to the studying of the gene expression.



Statistical analysis
 
The SPSS 20 software was used to analyze all data. The non-parametric tests (Kruskal-Wallis test) and (Mann-Whitney test) were used for comparison between the gene’s relative abundance of the (IVF) bovine embryos and both (SCNT) cloned embryos, P-values of less than 0.05 were considered significant.

RESULTS AND DISCUSSION

The expression of each studied gene (OCT4, DNMT1, BAX, GLUT1 and HSP70) was assessed in the early cleavage stages of the three types of embryos; this gene expression was compared between the three types of embryos. There was an increase in the gene expression for the genes OCT4, DNMT1, GLUT1 and HSP70 in the SCNT-B embryo compared to the other embryos (Fig 2), while the expression of the BAX gene, was high in both types of embryos IVF and SCNT-O compared to SCNT-B embryo (Fig 2), but these differences non-significant (P> 0.05). The expression of each studied gene (OCT4, DNMT1, BAX, GLUT1 and HSP70) was assessed in the late cleavage stage of the three types of embryos, this gene expression was compared between the three types of embryos. There was an increase in the gene expression for all genes OCT4, DNMT1, BAX, GLUT1 and HSP70 in the SCNT-B embryo compared to the other embryos, but these differences were non-significant (P>0.05) (Fig 3).

Fig 2: Comparison of expression of the studied genes in the early cleavage stage of the embryos produced by IVF, SCNT-B and SCNT-O.



​

Fig 3: Comparison of expression of the studied genes in the late cleavage stage of the embryos produced by IVF, SCNT-B and SCNT-O.



The study’s focus genes were chosen because they have been demonstrated to become transcriptionally active during embryonic genome activation (EGA) and participate in important biological processes during pre-implantation development (Ross et al., 2010). It could be feasible to obtain a further understanding of the reprogramming process and the gene expression patterns of preimplantation embryos by studying the gene expression before and after the maternal to-embryonic transition (Camargo et al., 2006), but the studies differed from each other in their results around the differences in the gene expression between the groups of embryos produced by the IVF or SCNT techniques and within the groups of SCNT.

The correct reprogramming which includes epigenetic modifications (DNA methylation and Histone modifications) is necessary for proper embryonic development and differentiation of cells (Cheng et al., 2019; Noh et al., 2016), suppressing sequences not intended to be expressed in the cell also constructs the fundamental gene expression infrastructure of the entire organism, such as various harmful parasitic and viral sequences and non-tissue-relevant genes found in the genome (Hall, 2013; Jeltsch et al., 2018), this regulation happen by the gene DNMT1. This results in the formation of various cell and tissue types through the control of certain gene expression patterns, which results in the states of gene expression being stable (Canovas et al., 2017; Dang and Zhang, 2019; Dor and Cedar, 2018). These epigenetic modifications are switches for the gene expression regulation (Hall, 2013; Marino-Ramirez et al., 2005; Van and Santos, 2018), so the abnormalities in gene expression return to the impaired transcriptional regulation mechanisms (Kishigami et al., 2006; Salehi et al., 2019).

Therefore, there may be an opposite interaction between the transporter glucose gene for energy that is important for embryo development GLUT1 and the apoptosis gene Bax, that means if the level expression of the GLUT1 gene is low causally related to the high incidence of apoptosis, from the other hand also there is an opposite interaction between the BAX gene of embryos and cytoprotective gene HSP70 causing increase the level expression of HSP70 gene to protects the embryos from the apoptosis caused by stress damage (Al-Malahi et al., 2022).  

Our results in (Fig 2 and 3) cleared that there were non-significant differences when we compared the groups of embryos (IVF, SCNT-B and SCNT-O) before and after the maternal-to-zygotic transition for each of the studied genes separately (OCT4, DNMT1, BAX, GLUT1 and HSP70), these findings consistent with the results of (Amarnath et al., 2007) whose compared the gene expression of genes profile included some like our studied genes (OCT4, DNMT1 and BAX ) between the IVF and SCNT buffalo embryos at the 8-cell stage and blastocyst and also agreed with EM et al., (2014) whose compared between the same embryos at the 8-cell stage for the genes (GLUT1, OCT4, DNMT1 and BAX). The insignificant differences between these types of embryos may be explained by the problems in the correct reprogramming of these genes or the quality of oocytes or embryos, the culture conditions, or the various modifications of the SCNT protocol like passage number of donor cells, source of donor cells, donor cell cycle stage and activation protocol, all of them can affect the gene expression embryonic patterns and the differences between the embryos produced by different reproductive techniques.

CONCLUSION

The transition from the maternal genome to the embryonic genome at the determined stage of development and the correct reprogramming and remodeling of the studied genes ensures balance in the genetic expression of these genes during the stages of embryonic development and this, in turn, ensures the continuation of high-quality embryonic development.

ACKNOWLEDGEMENT

The work at King Saud University, Riyadh, Saudi Arabia was funded by the Researcher Support Project (RSP-2025/R97), which the authors gratefully recognize. We would also like to express our gratitude to the Central Slaughterhouse in Riyadh for their assistance in gathering samples of bovine ovary.
 
Author contributions
 
All authors participate equally.
 
Institutional review board statement
 
Not applicable.
 
Informed consent statement
 
Informed consent was obtained from all subjects involved in the study.
 
Data availability statement
 
Not applicable.

CONFLICTS OF INTEREST

The authors declare no conflict of interest.   
 

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