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Research article
kwan-sik Min
Hankyong National University
Corresponding Author
ORCiD: https://orcid.org/0000-0002-5451-3085
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Equine chorionic gonadotropin (eCG) induces super-ovulation in laboratory animals. Notwithstanding its extensive usage, limited information is available regarding the differences between the in vivo effects of natural eCG (N-eCG) and recombinant eCG (R-eCG). This study aimed to investigate the gene expression profiles of mouse ovaries upon stimulation with N-eCG and R-eCG produced from CHO-suspension (CHO-S) cells. R-eCG gene was constructed and transfected into CHO-S cells and quantified. Subsequently, we determined the metabolic clearance rate (MCR) of N-eCG and R-eCG up to 24 h after intravenous administration through the mice tail vein and identified differentially expressed genes in both ovarian tissues, via quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC).
Results: R-eCG was markedly expressed initially after transfection and maintained until recovery on day 9. Glycan chains were substantially modified in R-eCG protein produced from CHO-S cells and eliminated through PNGase F treatment.
The MCR was higher for R-eCG than for N-eCG, and no significant difference was observed after 60 min. Notwithstanding their low concentrations, R-eCG and N-eCG were detected in the blood at 24h post-injection. Microarray analysis of ovarian tissue revealed that 20 of 12,816 genes assessed therein were significantly up-regulated and 43 genes were down-regulated by >2-fold in the group that received R-eCG (63 [0.49%] differentially regulated genes in total). The microarray results were concurrent with and hence validated by those of RT-PCR, qRT-PCR, and IHC analyses.
Conclusions: The present results indicate that R-eCG can be adequately produced through a cell-based expression system through post-translational modification of eCG and can induce ovulation in vivo. These results provide novel insights into the molecular mechanisms underlying the up- or down-regulation of specific ovarian genes and the production of R-eCG with enhanced biological activity in vivo.
Keywords
R-eCG, CHO-S cells, MCR, microarray, qRT-PCR, immunohistochemistry
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Figure 3
Figure 4
Figure 5
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This is a list of supplementary files associated with this preprint. Click to download.
- MinTableword200824.pptx
- SupplemtaryTable1andFig.1.2200824.ppt
Additional file 1: Table S1. List of primers used for RT-PCR and qRT-PCR. Fourteen genes from different categories were chosen for RT-PCR and qRT-PCR analyses. The gene for -actin was used as the endogenous control. Additional file 2: Figure S1. Gene ontology of biological processes and molecular functions. Genes distribution of >2-fold differentially expressed genes between N-eCG and R-eCG. Additional file 3: Figure S2. Gene ontology of biological processes and molecular functions. Gene ontology pie diagram of >2-fold differentially expressed genes between N-eCG and R-eCG-treated ovaries. The up-regulated or down-regulated genes are categorized by the GO term “biological process and molecular function”.
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On 23 Aug, 2020
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On 23 Aug, 2020
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Editorial decision: Major revision
On 11 Aug, 2020
Review #3 received
Received 25 Jul, 2020
Review #2 received
Received 25 Jul, 2020
Reviewer #3 agreed
On 13 Jul, 2020
Reviewer #2 agreed
On 09 Jul, 2020
Review #1 received
Received 15 Jun, 2020
Reviewer #1 agreed
On 12 Jun, 2020
Reviewers invited
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See the published version of this preprint at BMC Biotechnology.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research article
kwan-sik Min
Hankyong National University
Corresponding Author
ORCiD: https://orcid.org/0000-0002-5451-3085
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
View the published article at BMC Biotechnology.
No community comments so far
Editorial decision: Accept
On 03 Nov, 2020
Editor assigned
On 31 Oct, 2020
Submission checks complete
On 31 Oct, 2020
Editor invited
On 31 Oct, 2020
No comments provided
Editor assigned
On 28 Oct, 2020
Submission checks complete
On 27 Oct, 2020
Editor invited
On 27 Oct, 2020
Version 4
Posted 08 Oct, 2020
No comments provided
Editorial decision: Minor revision
On 21 Oct, 2020
Editor assigned
On 05 Oct, 2020
Editor invited
On 04 Oct, 2020
Submission checks complete
On 04 Oct, 2020
Version (no preprint)
Posted 08 Oct, 2020
Editorial decision: Minor revision
On 25 Oct, 2020
Editor assigned
On 23 Oct, 2020
Submission checks complete
On 22 Oct, 2020
Editor invited
On 22 Oct, 2020
This version was not preprinted
No comments provided
Editorial decision: Minor revision
On 29 Sep, 2020
Review #1 received
Received 21 Sep, 2020
Reviewer #2 agreed
On 18 Sep, 2020
Review #2 received
Received 18 Sep, 2020
Reviewers invited
Invitations sent on 15 Sep, 2020
Reviewer #1 agreed
On 15 Sep, 2020
Editor assigned
On 04 Sep, 2020
Submission checks complete
On 03 Sep, 2020
Editor invited
On 03 Sep, 2020
No comments provided
Editorial decision: Revise before sending to peer reviewers
On 25 Aug, 2020
Editor assigned
On 24 Aug, 2020
Submission checks complete
On 23 Aug, 2020
Editor invited
On 23 Aug, 2020
No comments provided
Editorial decision: Major revision
On 11 Aug, 2020
Review #3 received
Received 25 Jul, 2020
Review #2 received
Received 25 Jul, 2020
Reviewer #3 agreed
On 13 Jul, 2020
Reviewer #2 agreed
On 09 Jul, 2020
Review #1 received
Received 15 Jun, 2020
Reviewer #1 agreed
On 12 Jun, 2020
Reviewers invited
Invitations sent on 10 Jun, 2020
Editor assigned
On 12 May, 2020
Submission checks complete
On 11 May, 2020
Editor invited
On 11 May, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Biotechnology and Bioengineering
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at BMC Biotechnology.
Background: Equine chorionic gonadotropin (eCG) induces super-ovulation in laboratory animals. Notwithstanding its extensive usage, limited information is available regarding the differences between the in vivo effects of natural eCG (N-eCG) and recombinant eCG (R-eCG). This study aimed to investigate the gene expression profiles of mouse ovaries upon stimulation with N-eCG and R-eCG produced from CHO-suspension (CHO-S) cells. R-eCG gene was constructed and transfected into CHO-S cells and quantified. Subsequently, we determined the metabolic clearance rate (MCR) of N-eCG and R-eCG up to 24 h after intravenous administration through the mice tail vein and identified differentially expressed genes in both ovarian tissues, via quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC).
Results: R-eCG was markedly expressed initially after transfection and maintained until recovery on day 9. Glycan chains were substantially modified in R-eCG protein produced from CHO-S cells and eliminated through PNGase F treatment.
The MCR was higher for R-eCG than for N-eCG, and no significant difference was observed after 60 min. Notwithstanding their low concentrations, R-eCG and N-eCG were detected in the blood at 24h post-injection. Microarray analysis of ovarian tissue revealed that 20 of 12,816 genes assessed therein were significantly up-regulated and 43 genes were down-regulated by >2-fold in the group that received R-eCG (63 [0.49%] differentially regulated genes in total). The microarray results were concurrent with and hence validated by those of RT-PCR, qRT-PCR, and IHC analyses.
Conclusions: The present results indicate that R-eCG can be adequately produced through a cell-based expression system through post-translational modification of eCG and can induce ovulation in vivo. These results provide novel insights into the molecular mechanisms underlying the up- or down-regulation of specific ovarian genes and the production of R-eCG with enhanced biological activity in vivo.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
This is a list of supplementary files associated with this preprint. Click to download.
- MinTableword200824.pptx
- SupplemtaryTable1andFig.1.2200824.ppt
Additional file 1: Table S1. List of primers used for RT-PCR and qRT-PCR. Fourteen genes from different categories were chosen for RT-PCR and qRT-PCR analyses. The gene for -actin was used as the endogenous control. Additional file 2: Figure S1. Gene ontology of biological processes and molecular functions. Genes distribution of >2-fold differentially expressed genes between N-eCG and R-eCG. Additional file 3: Figure S2. Gene ontology of biological processes and molecular functions. Gene ontology pie diagram of >2-fold differentially expressed genes between N-eCG and R-eCG-treated ovaries. The up-regulated or down-regulated genes are categorized by the GO term “biological process and molecular function”.
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