Single-cell DNA methylation sequencing reveals epigenetic alterations in mouse oocytes superovulated with different dosages of gonadotropins

Abstract

Background: Epigenetic abnormalities caused by superovulation have recently attracted increasing attention. Superovulation with exogenous hormones may prevent oocytes from establishing an appropriate epigenetic state, and this effect may extend to the methylation programming in preimplantation embryos, as de novo DNA methylation is a function of developmental stage of follicles and oocyte size. Follicle-stimulating hormone (FSH) and human menopausal gonadotropin (hMG) are common gonadotropins used for superovulation, and appropriate concentrations of these gonadotropins might be necessary. However, no systematic study on the effects of DNA methylation alterations in oocytes associated with superovulation with different dosages of FSH/hMG at the singlecell level has yet been reported. In the current study, different dosages of FSH/hMG combined with human chorionic gonadotropin (hCG) were used in female mice to generate experimental groups, while naturally matured oocytes and oocytes superovulated with only hCG were respectively used as controls. Single-cell level DNA methylation sequencing was carried out on all these matured oocytes.Results: In this study, we revealed that the genome-wide methylation pattern and CG methylation level of the maternal imprinting control regions of all mature oocytes were globally conserved and stable. However, methylation alterations associated with superovulation were found at a specific set of loci, and the differentially methylated regions (DMRs) mainly occurred in regions other than promoters. Furthermore, some of the annotated genes in the DMRs were involved in biological processes such as glucose metabolism, nervous system development, cell cycle, cell proliferation, and embryo implantation and were altered in all dosages of FSH/hMG group (for example, Gfod2 and SYF2). Other genes were impaired only after high gonadotropin dosages (for instance, Sox17 and Phactr4).

The Proteome, Not the Transcriptome, Predicts that Oocyte Superovulation Affects Embryonic Phenotypes in Mice

“…Instead we propose a model whereby the superovulation treatment stops the oocytes prematurely on their way to attain the proper size and natural quality. A similar proposal has been made in an independent study, on the basis of DNA methylation data in superovulated vs. naturally ovulated mouse oocytes 27 .…”

“…If the above model is correct, then the logical follow-up experiment is to allow for more time until the release of the oocytes from the ovary, expecting to see a relief of the superovulation-associated deficits. Unlike the gonadotropin dosage 27,79,80 and the time of gonadotropin stimulation in relation to estrous cycle 81 and photoperiod 82 , the interval of the gonadotropins has not received much consideration, although it goes by itself that the accumulation of gene products in oocytes is a function of time during oogenesis. Compared to the natural length of the ovarian cycle in F1 mice, which is 4 days 83 , the interval eCG-hCG of the conventional superovulation spans only 2 days, thereby triggering the idea to assess the extent of preovulatory maturation attained until the administration of hCG.…”

“…Likewise, while some studies linked the superovulation to a direct effect on the acquisition of DNA methylation in oocytes [19][20][21][22][23] , including epimutations 24 , other studies found no such effect 25,26 . In particular, one study suggested that superovulation results in immature oocytes that have not completed the acquisition of DNA methylation 27 , whereas another study found that oocyte diameters -which correlate with oocyte maturity and DNA methylation 28 -do not differ between superovulation and natural ovulation 26 . In most of the studies above, there was a common confounder: the time spent by the oocytes in the genital tract of the stimulated female (interval between superovulation and collection for study).…”

Assessment of Cardiovascular Health of Children Ages 6 to 10 Years Conceived by Assisted Reproductive Technology

“… 39 , 40 These epigenetic alterations may be associated with the infertility backgrounds of parents, superovulation, or factors associated with the ART procedure. 41 , 42 The alterations may persist into childhood and be associated with worsened cardiovascular health among offspring. 33 , 34 Second, increased oxidative stress (OS) may also be associated with altered cardiac structure and function among offspring conceived by ART.…”

Embryo culture media differentially alter DNA methylating enzymes and global DNA methylation in embryos and oocytes

“…They also added that DNA methylation of superovulated oocytes obtained from prepubertal mice differs from the that of adult oocytes, while oocytes from superovulated adult females or naturally ovulated oocytes show slight differences. Similarly, Huo et al (Huo et al 2020 ) analyzed the effect of different dosages of FSH/hMG combined with hCG in female mice and found that superovulation causes methylation alterations at a specific set of loci, and the differentially methylated regions mainly occurred in regions other than promoters many of which are involved in vital biological processes such as glucose metabolism, nervous system development, cell cycle, cell proliferation, and embryo implantation. In the current study, we did not find any differences between IV group, in which mice were hormonally (5 IU PMSG and HCG) stimulated, and Vas group, not hormonally stimulated, in Dnmt1 and 3a and also global DNA methylation in MII stage oocytes.…”