High Dosages of Equine Chorionic Gonadotropin Exert Adverse Effects on the Developmental Competence of IVF-Derived Mouse Embryos and Cause Oxidative Stress-Induced Aneuploidy

Abstract

Gonadotropins play vital roles in the regulation of female reproductive ability and fertility. Our study aimed to determine the effects of superovulation induced by increasing doses of equine chorionic gonadotropin [eCG; also referred to as pregnant mare serum gonadotropin (PMSG)] on the developmental competence of mouse embryos and on aneuploidy formation during in vitro fertilization (IVF). eCG dose-dependently enhanced the oocyte yield from each mouse. Administration of 15 IU eCG significantly reduced the fertilization rate and the formation of four-cell embryos and blastocysts and increased the risk of chromosome aneuploidy. The IVF-derived blastocysts in the 15 IU eCG treatment group had the fewest total cells, inner cell mass (ICM) cells and trophectoderm (TE) cells. Moreover, more blastocysts and fewer apoptotic cells were observed in the 0, 5, and 10 IU eCG treatment groups than in the 15 IU eCG treatment group. We also investigated reactive oxygen species (ROS) levels and variations in several variables: mitochondrial membrane potential (MMP); active mitochondria; mitochondrial superoxide production; adenosine triphosphate (ATP) content; spindle structures; chromosome karyotypes; microfilament distribution; and the expression of Aurora B [an important component of the chromosomal passenger complex (CPC)], the spindle assembly checkpoint (SAC) protein mitotic arrest deficient 2 like 1 (MAD2L1), and the DNA damage response (DDR) protein γH2AX. Injection of 15 IU eCG increased ROS levels, rapidly reduced MMP, increased active mitochondria numbers and mitochondrial superoxide production, reduced ATP content, increased abnormal spindle formation rates, and induced abnormalities in chromosome number and microfilament distribution, suggesting that a high dose of eCG might alter developmental competence and exert negative effects on IVF-obtained mouse embryos. Additionally, the appearance of γH2AX and the significantly increased expression of Aurora B and MAD2L1 suggested that administration of relatively high doses of eCG caused Aurora B-mediated SAC activation triggered by ROS-induced DNA damage in early mouse IVF-derived embryos for self-correction of aneuploidy formation. These findings improve our understanding of the application of gonadotropins and provide a theoretical basis for gonadotropin treatment.

The proteome, not the transcriptome, predicts that oocyte superovulation affects embryonic phenotypes in mice

“…At the blastocyst stage, we recorded a depletion of the inner cell mass under the same gonadotropin dosage (10 I.U.) as used in another study 15 , although the mouse strains were different. The depletion of epiblast and primitive endoderm cells echoes our previous work 71 , in which we reported on a small – but not negligible – proportion of non-manipulated, in vivo-produced blastocysts lacking epiblast cells in their inner cell masses.…”

“…Superovulated oocytes had reduced ability for endocytosis 11 and derivative blastocysts had fewer microvilli 12 than naturally ovulated counterparts, but ultrastructural differences were otherwise unimpressive. Some studies reported an increase of the rate of embryo aneuploidy and fetal malformation by use of gonadotropins [13][14][15][16][17] , while others contradicted these findings 4,18,19 . Some studies linked the superovulation to a direct effect on the acquisition of DNA methylation in oocytes [20][21][22][23][24] , including epimutations 25 , while…”

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

“…At the blastocyst stage, we recorded a depletion of the inner cell mass under the same gonadotropin dosage (10 I.U.) as used in another study 14 , although the mouse strains were different. The depletion of epiblast and primitive endoderm cells echoes our previous work 72 , in which we reported on a small -but not negligible -proportion of non-manipulated, in vivo-produced blastocysts lacking epiblast cells in their inner cell masses.…”

“…Superovulated mouse oocytes had reduced ability for endocytosis 10 and derivative blastocysts had fewer microvilli 11 than naturally ovulated counterparts, but ultrastructural differences were otherwise unimpressive. Some studies reported an increase of the rate of embryo aneuploidy and fetal malformation by use of gonadotropins [12][13][14][15][16] while others contradicted these findings 4,17,18 . 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 .…”

Impact of Superovulation And In Vitro Fertilization On LINE-1 Copy Number And Telomere Length In C57BL/6J Mice Blastocysts

“…Barbara McClintock's Nobel Prizing winning research previously identi ed activation of retrotransposons as a response to stress [37]. A derivative hypothesis is that stress from ovarian hyperstimulation [38][39][40] and/or embryo culture activates transcription of L1. Our ndings that prolonged embryo culture increased L1 copy number are consistent with Professor McClintock's model.…”