Abstract
Maternal hyperhomocysteinemia (HCY) induced by genetic defects in methionine cycle enzymes or vitamin imbalance is known to be a pathologic factor that can impair embryonal brain development and cause long-term consequences in the postnatal brain development as well as changes in the expression of neuronal genes. Studies of the gene expression on this model requires the selection of optimal housekeeping genes. This work aimed to analyze the expression stability of housekeeping genes in offspring brain. Pregnant female Wistar rats were treated daily with a 0.15% L-methionine solution in the period starting on the 4th day of pregnancy until delivery, to cause the increase in the homocysteine level in fetus blood and brain. Housekeeping gene expression was assessed by RT-qPCR on whole embryonic brain and selected rat brain areas at P20 and P90. The amplification curves were analyzed, and raw means Cq data were imported to the RefFinder online tool to assess the reference genes stability. Most of the analyzed genes showed high stability of mRNA expression in the fetal brain at both periods of analysis (E14 and E20). However, the most stably expressed genes at different age points differed. Actb, Ppia, Rpl13a are the most stably expressed on E14, Ywhaz, Pgk1, Hprt1 – on E20 and P20, Hprt1, Actb, and Pgk1 – on P90. Gapdh gene used as a reference in various studies demonstrates high stability only in the hippocampus and cannot be recommended as the optimal reference gene on HCY model. Hprt1 and Pgk1 genes were found to be the most stably expressed in the brain of rat subjected to HCY. These two genes showed high stability in the brain on E20 and in various areas of the brain on the P20 and P90. On E14, the preferred genes for normalization are Actb, Ppia, Rpl13a.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data Availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Code Availability
STATISTICA 10.0 (Statsoft).
Abbreviations
- HHC:
-
Hyperhomocysteinemia
References
Althausen S, Paschen W (2000) Homocysteine-induced changes in mRNA levels of genes coding for cytoplasmic- and endoplasmic reticulum-resident stress proteins in neuronal cell cultures. Mol Brain Res 84:32–40. https://doi.org/10.1016/S0169-328X(00)00208-4
Arutjunyan AV, Milyutina YP, Shcherbitskaia AD et al (2020) Neurotrophins of the fetal brain and placenta in prenatal hyperhomocysteinemia. Biochem 85:213–223. https://doi.org/10.1134/S000629792002008X
Baydas G, Koz ST, Tuzcu M et al (2007) Effects of maternal hyperhomocysteinemia induced by high methionine diet on the learning and memory performance in offspring. Int J Dev Neurosci 25:133–139. https://doi.org/10.1016/j.ijdevneu.2007.03.001
Baydas G, Koz ST, Tuzcu M, Nedzvetsky VS (2008) Melatonin prevents gestational hyperhomocysteinemia-associated alterations in neurobehavioral developments in rats. J Pineal Res 44:181–188. https://doi.org/10.1111/j.1600-079X.2007.00506.x
Blaise SA, Nédélec E, Schroeder H et al (2007) Gestational vitamin B deficiency leads to homocysteine-associated brain apoptosis and alters neurobehavioral development in rats. Am J Pathol 170:667–679. https://doi.org/10.2353/ajpath.2007.060339
Boldyrev AA (2009) Molecular mechanisms of homocysteine toxicity. Biochem 74:589–598. https://doi.org/10.1134/S0006297909060017
Bonefeld BE, Elfving B, Wegener G (2008) Reference genes for normalization: a study of rat brain tissue. Synapse 62:302–309. https://doi.org/10.1002/syn.20496
Bustelo M, Bruno MA, Loidl CF et al (2020) Statistical differences resulting from selection of stable reference genes after hypoxia and hypothermia in the neonatal rat brain. PLoS ONE 15:e0233387. https://doi.org/10.1371/journal.pone.0233387
Chapman JR, Waldenström J (2015) With reference to reference genes: a systematic review of endogenous controls in gene expression studies. PLoS ONE 10:e0141853. https://doi.org/10.1371/journal.pone.0141853
Hamelet J, Demuth K, Paul J-L et al (2007) Hyperhomocysteinemia due to cystathionine beta synthase deficiency induces dysregulation of genes involved in hepatic lipid homeostasis in mice. J Hepatol 46:151–159. https://doi.org/10.1016/j.jhep.2006.07.028
Jiang T, Dai S, Yi Y et al (2020) The combination of hprt and gapdh is the best compound reference genes in the fetal rat hippocampus. Dev Neurobiol 80:229–238. https://doi.org/10.1002/dneu.22779
Koz ST, Gouwy NT, Demir N et al (2010) Effects of maternal hyperhomocysteinemia induced by methionine intake on oxidative stress and apoptosis in pup rat brain. Int J Dev Neurosci 28:325–329. https://doi.org/10.1016/j.ijdevneu.2010.02.006
Luo X, Xiao L, Yang H et al (2014) Homocysteine downregulates gene expression of heme oxygenase-1 in hepatocytes. Nutr Metab (Lond) 11:55. https://doi.org/10.1186/1743-7075-11-55
Paxinos G, Watson C (2007) The rat brain in stereotaxic coordinates. Elsevier
Postnikova TY, Amakhin DV, Trofimova AM et al (2022) Maternal hyperhomocysteinemia produces memory deficits associated with impairment of long-term synaptic plasticity in young rats. Cells 12:58. https://doi.org/10.3390/cells12010058
Schwarz AP, Kovalenko AA, Malygina DA et al (2020a) Reference gene validation in the brain regions of young rats after pentylenetetrazole-induced seizures. Biomedicines 8. https://doi.org/10.3390/BIOMEDICINES8080239
Schwarz AP, Malygina DA, Kovalenko AA et al (2020b) Multiplex qPCR assay for assessment of reference gene expression stability in rat tissues/samples. Mol Cell Probes 53:101611. https://doi.org/10.1016/j.mcp.2020.101611
Schwarz AP, Dyomina AV, Zakharova MV et al (2022) The reference gene validation in the brain of rats during antioxidant and anti-inflammatory treatment in the lithium-pilocarpine model of temporal epilepsy. J Evol Biochem Physiol 58:930–940. https://doi.org/10.1134/S0022093022030279
Shcherbitskaia AD, Vasilev DS, Milyutina YP et al (2020) Maternal hyperhomocysteinemia induces neuroinflammation and neuronal death in the rat offspring cortex. Neurotox Res 38:408–420. https://doi.org/10.1007/s12640-020-00233-w
Shcherbitskaia AD, Vasilev DS, Milyutina YP et al (2021) Prenatal hyperhomocysteinemia induces glial activation and alters neuroinflammatory marker expression in infant rat hippocampus. Cells 10. https://doi.org/10.3390/cells10061536
Shcherbitskaia AD, Kovalenko AA, Milyutina YP, Vasilev DS (2022) Thyroid hormone production and transplacental transfer in the mother–fetus system during gestational hyperhomocysteinemia. Neurochem J 16:249–262. https://doi.org/10.1134/S1819712422030102
Tian X, Gong L, Jin A et al (2019) E3 ubiquitin ligase siah–1 nuclear accumulation is critical for homocysteine–induced impairment of C6 astroglioma cells. Mol Med Rep. https://doi.org/10.3892/mmr.2019.10449
Tsitsiou E, Sibley CP, D’Souza SW et al (2011) Homocysteine is transported by the microvillous plasma membrane of human placenta. J Inherit Metab Dis 34:57–65. https://doi.org/10.1007/s10545-010-9141-3
Xie F, Xiao P, Chen D et al (2012) miRDeepFinder: a miRNA analysis tool for deep sequencing of plant small RNAs. Plant Mol Biol 80:75–84. https://doi.org/10.1007/s11103-012-9885-2
Yakovlev AV, Kurmashova E, Zakharov A, Sitdikova GF (2018) Network-driven activity and neuronal excitability in hippocampus of neonatal rats with prenatal hyperhomocysteinemia. Bionanoscience 8:304–309. https://doi.org/10.1007/s12668-017-0450-y
Yakovleva O, Bogatova K, Mukhtarova R et al (2020) Hydrogen sulfide alleviates anxiety, motor, and cognitive dysfunctions in rats with maternal hyperhomocysteinemia via mitigation of oxidative stress. Biomolecules 10:995. https://doi.org/10.3390/biom10070995
Zhang JW, Yan R, Tang YS et al (2017) Hyperhomocysteinemia-induced autophagy and apoptosis with downregulation of hairy enhancer of split 1/5 in cortical neurons in mice. Int J Immunopathol Pharmacol 30:371–382. https://doi.org/10.1177/0394632017740061
Zhou J, Zhang X, Ren J et al (2016) Validation of reference genes for quantitative real-time PCR in valproic acid rat models of autism. Mol Biol Rep 43:837–847. https://doi.org/10.1007/s11033-016-4015-x
Funding
This work was supported by the Russian Science Foundation (№22-15-00393).
Author information
Authors and Affiliations
Contributions
All authors contributed to the study conception and design. Animal experiments were performed by A. D. S. and A. V. M. Material preparation, data collection and analysis were performed by A. A. K. The first draft of the manuscript was written by A. A. K., and D. S. V. Writing review and editing were performed by A. P. S., A. D. S. and A. V. A.; General supervision and secured funding for this project were performed by A. V. A. All authors read and approved the final manuscript.
Corresponding author
Ethics declarations
Competing Interests
The authors declare no competing interests.
Ethics Approval
All experiments were performed with permission of the Ethics Committee of D.O. Ott Institute of Obstetrics, Gynecology and Reproductology and according to the European Communities Council Directive #86/609 for the Care of Laboratory Animals.
Consent to Participate
Not applicable.
Consent for Publication
Not applicable.
Additional information
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic Supplementary Material
Below is the link to the electronic supplementary material.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Kovalenko, A.A., Schwarz, A.P., Shcherbitskaia, A.D. et al. Reference Gene Validation in the Embryonic and Postnatal Brain in the Rat Hyperhomocysteinemia Model. Neurotox Res 42, 19 (2024). https://doi.org/10.1007/s12640-024-00698-z
Received:
Revised:
Accepted:
Published:
DOI: https://doi.org/10.1007/s12640-024-00698-z