Dysregulated Gene Expression of Imprinted and X-Linked Genes: A Link to Poor Development of Bovine Haploid Androgenetic Embryos

Abstract

Mammalian uniparental embryos are efficient models for genome imprinting research and allow studies on the contribution of the paternal and maternal genomes to early embryonic development. In this study, we analyzed different methods for production of bovine haploid androgenetic embryos (hAE) to elucidate the causes behind their poor developmental potential. Results indicate that hAE can be efficiently generated by using intracytoplasmic sperm injection and oocyte enucleation at telophase II. Although androgenetic haploidy does not disturb early development up to around the 8-cell stage, androgenetic development is disturbed after the time of zygote genome activation and hAE that reach the morula stage are less capable to reach the blastocyst stage of development. Karyotypic comparisons to parthenogenetic- and ICSI-derived embryos excluded chromosomal segregation errors as causes of the developmental constraints of hAE. However, analysis of gene expression indicated abnormal levels of transcripts for key long non-coding RNAs involved in X chromosome inactivation and genomic imprinting of the KCNQ1 locus, suggesting an association with X chromosome and some imprinted loci. Moreover, transcript levels of methyltransferase 3B were significantly downregulated, suggesting potential anomalies in hAE establishing de novo methylation. Finally, the methylation status of imprinted control regions for XIST and KCNQ1OT1 genes remained hypomethylated in hAE at the morula and blastocyst stages, confirming their origin from spermatozoa. Thus, our results exclude micromanipulation and chromosomal abnormalities as major factors disturbing the normal development of bovine haploid androgenotes. In addition, although the cause of the arrest remains unclear, we have shown that the inefficient development of haploid androgenetic bovine embryos to develop to the blastocyst stage is associated with abnormal expression of key factors involved in X chromosome activity and genomic imprinting.

Haploid androgenetic development in bovines reveals imbalanced WNT signaling and impaired cell fate differentiation

“…On the other hand, as previously reported (Aguila et al, 2021;Hu et al, 2015;Latham et al, 1994;Ogawa et al, 2006;Sotomaru et al, 2001), we report perturbed imprinting expression patterns during uniparental development with the overexpression of maternally expressed imprinted genes in parthenotes and overexpression of paternally expressed imprinted genes in androgenotes, consistent with the notion that the absence of the reciprocal parental allele leads to the overexpression of imprinted genes. This differential imprinting expression pattern of haploid embryos determines not only the parental origin of the embryo (Daughtry & Mitalipov, 2014;Sritanaudomchai et al, 2010) but also its association with developmental outcomes (Aguila et al, 2021;Bos-Mikich et al, 2016;Latham et al, 2002). For instance, embryos with only the maternal genome show altered expression patterns of key enzymes required for epigenetic reprogramming (Aguila et al, 2021;Kono, 2006;Peng et al, 2015;Sagi et al, 2019) In a similar fashion, the bioinformatic analysis revealed a differential profile of pluripotency factors, particularly an unbalanced WNT pathway of overrepresented canonical factors.…”

“…In mice (Ogawa et al, 2006) and humans (Sagi et al, 2019) androgenetic blastocysts have a lower number of cells and are associated with hindered blastulation. Our previous study also reported a poor blastulation rate (26%) for hAE compared to biparental (80%) or hPE (68%) (Aguila et al, 2021). Notably here, GSK3B inhibition enhanced embryonic competence by increasing the "blastulation rate", seen as the proportion of morulas becoming blastocysts.…”

“…This differential imprinting expression pattern of haploid embryos determines not only the parental origin of the embryo (Daughtry & Mitalipov, 2014;Sritanaudomchai et al, 2010) but also its association with developmental outcomes (Aguila et al, 2021;Bos-Mikich et al, 2016;Latham et al, 2002). For instance, embryos with only the maternal genome show altered expression patterns of key enzymes required for epigenetic reprogramming (Aguila et al, 2021;Kono, 2006;Peng et al, 2015;Sagi et al, 2019) In a similar fashion, the bioinformatic analysis revealed a differential profile of pluripotency factors, particularly an unbalanced WNT pathway of overrepresented canonical factors. In the bovine species, modulation of the activity of WNT signaling is necessary for development of ESC (Bogliotti et al, 2018;Xiao, Sosa, et al, 2021).…”

Simple and Efficient Chemically Defined In Vitro Maturation and Embryo Culture System for Bovine Embryos

“…Cell count in the blastocyst is associated with embryo quality, resistance to cryopreservation, and ultimately with pregnancy and deliveries after transfer [ 14 , 72 , 73 , 74 ]. The evaluation of the total number of cells showed that the embryos generated under defined conditions showed a quality similar to that obtained in the presence of FBS/BSA.…”

Preimplantation Developmental Competence of Bovine and Porcine Oocytes Activated by Zinc Chelation

“…Here, we provide the first insights into parthenogenetic blastocyst development in cattle using Zn chelation. In this species, assisted oocyte activation has been used to generate haploid and diploid parthenogenetic [ 28 ] and androgenetic embryos [ 29 ]. Moreover, it is an essential step in SCNT and for assisting round spermatid sperm injection.…”