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Epiblast production and thereby totipotency of the first two blastomeres is balanced or unbalanced depending on the region where the oocyte was fertilized

ABSTRACT: Following fertilization in mice and humans, the first two blastomeres are not equivalent but one produces more epiblast than the other, so that they do not feature equal totipotency. Research into the causes has overlooked that the epiblast imbalance is preceded by a fertilization imbalance, since in nature the spermatozoon fertilizes the oocyte preferentially in the animal hemisphere. We conceived a hypothesis that the two imbalances are linked to each other, and broke it down into testable predictions. If the two imbalances were interdependent, then changing the site of sperm entry in the oocyte should change the extent of epiblast imbalance. Thus, we evened out the fertilization imbalance, using intracytoplasmic sperm injection (ICSI) to fertilize mouse oocytes also in the vegetal hemisphere as well as in the cortex around the animal-vegetal midline (equator). Resultant embryos were bisected at the 2-cell stage, and the twin blastocysts originated from the sister blastomeres were analyzed. Against similarity in mRNA levels of epiblast genes, twin blastocysts differed in function, as measured by NANOG protein expression and derivation of embryonic stem cells, and the epiblast imbalance was greater after oocyte fertilization at the equator. There is no simple way to explain the positional effect other than through differences in molecular composition of the ooplasm, which, moreover, should also be apportioned variably at first zygotic division. We tested these predictions by measuring the orientation of first zygotic division with respect to the ICSI site; and by measuring the composition of bisected oocytes’ hemispheres using half-cell proteomics. Since the hemispheres have different compositions depending on the bisection axis, and since the angle of the first division is variable, we propose that variable partition of non-homogeneous ooplasm sets the stage for the epiblast imbalance. These results attest to the architectural influence of maternal factors on mammalian embryogenesis.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Early Embryonic Cell, Oocyte

SUBMITTER: Hannes Drexler

LAB HEAD: Hannes C. A. Drexler

PROVIDER: PXD067319 | Pride | 2026-01-19

REPOSITORIES: Pride

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			Action	DRS
	20250626_1366_Michele_half-Oocytes_Jena25_oocDIA60min_01_Slot2-3_1_6456.d.7z	Other
	20250626_1366_Michele_half-Oocytes_Jena25_oocDIA60min_02_Slot2-4_1_6457.d.7z	Other
	20250626_1366_Michele_half-Oocytes_Jena25_oocDIA60min_03_Slot2-5_1_6458.d.7z	Other
	20250626_1366_Michele_half-Oocytes_Jena25_oocDIA60min_04_Slot2-6_1_6459.d.7z	Other
	20250626_1366_Michele_half-Oocytes_Jena25_oocDIA60min_05_Slot2-7_1_6460.d.7z	Other

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Project description:The present study was undertaken to discover molecular markers in bovine cumulus cells predictive of oocyte competence and elucidate their functional significance. Differences in RNA transcript abundance in cumulus cells harvested from oocytes of adult versus prepubertal animals (model of poor oocyte quality) were identified by microarray analysis. Four genes of interest encoding for the lysosomal cysteine proteinases cathepsin B, S, K and Z and displaying greater transcript abundance in cumulus cells surrounding oocytes harvested from prepubertal animals were chosen for further investigation. Greater mRNA abundance for such genes in cumulus cells of prepubertal oocytes was confirmed by real time RT-PCR. Elevated transcript abundance for cathepsins B, S and Z was also observed in cumulus cells surrounding adult metaphase II oocytes that developed to the blastocyst stage at a low percentage following parthenogenetic activation, versus those that developed at a high percentage. Functional significance of cumulus cell cathepsin expression to oocyte competence was confirmed by treatment of cumulus oocyte complexes during in vitro oocyte maturation with a cell permeable cysteine proteinase (cathepsin) inhibitor. Inhibitor treatment decreased apoptotic nuclei in the cumulus layer and enhanced development of parthenogenetically activated and in vitro fertilized adult oocytes to the blastocyst stage. Stimulatory effects of inhibitor treatment during meiotic maturation on subsequent embryonic development were not observed when oocytes were matured in the absence of cumulus cells. Results support a functional role for cumulus cell cathepsins in compromised oocyte competence and suggest that cumulus cell cathepsin mRNA abundance may be predictive of oocyte quality. Keywords: Bovine, microarray, cDNA, cumulus cells Differences in RNA transcript abundance in cumulus cells harvested from oocytes of adult versus prepubertal animals (model of poor oocyte quality) were identified by microarray analysis. Total RNA from pools of cumulus cells (n = 4) collected from adult and prepubertal animals for microarray experiments was amplified. Two color microarray experiments were conducted using a bovine cDNA array containing expressed sequence tags (ESTs) representing approximately 15200 unique genes. Hybridizations were performed on duplicate slides (prepubertal versus adult) and incorporated a dye swap. The total number of slides used is eight.

Project description:The identification of genes and deduced pathways from the mature human oocyte can help us better understand oogenesis, folliculogenesis, fertilization, and embryonic development. Human metaphase II oocytes were used within minutes after removal from the ovary, and its transcriptome was compared with a reference sample consisting of a mixture of total RNA from 10 different normal human tissues not including the ovary. RNA amplification was performed by using a unique protocol. Affymetrix Human Genome U133 Plus 2.0 GeneChip arrays were used for hybridizations. Compared with reference samples, there were 5,331 transcripts significantly up-regulated and 7,074 transcripts significantly down-regulated in the oocyte. Of the oocyte up-regulated probe sets, 1,430 have unknown function. A core group of 66 transcripts was identified by intersecting significantly up-regulated genes of the human oocyte with those from the mouse oocyte and from human and mouse embryonic stem cells. GeneChip array results were validated using RT-PCR in a selected set of oocyte-specific genes. Within the up-regulated probe sets, the top overrepresented categories were related to RNA and protein metabolism, followed by DNA metabolism and chromatin modification. This report provides a comprehensive expression baseline of genes expressed in in vivo matured human oocytes. Further understanding of the biological role of these genes may expand our knowledge on meiotic cell cycle, fertilization, chromatin remodeling, lineage commitment, pluripotency, tissue regeneration, and morphogenesis. Experiment Overall Design: We generated a database of the human oocyte transcriptome by comparing the transcripts in the oocyte with the reference samples, which contain mRNA from several somatic tissues. Experiment Overall Design: Human oocytes were obtained from three patients undergoing an assisted reproductive treatment (ART) at the Unit of Reproductive Medicine of Clinica Las Condes, Santiago, Chile. It is important to emphasize that the routine in vitro fertilization protocol at Clinica Las Condes calls for fertilizing only those oocytes that will be transferred into the uterus of the patient. Therefore, there is always a surplus of oocytes. We then had the opportunity to use specific criteria to select donors as follows: (i) <35 years old, (ii) reproductively healthy with regular ovulatory cycles, (iii) male factor as the only cause of infertility, and (iv) considerable number of developing follicles that assured spared oocytes. The experimental protocol was reviewed and approved by a local independent Ethics Review Board. All donors signed informed consent. At the time this manuscript was submitted, all three donors had already conceived; two of them got pregnant during the ART cycle in which our samples were collected, and the third one got pregnant after a spontaneous cycle with artificial insemination using donated sperm. Ovarian stimulation, oocyte retrieval, and cell lysis were performed as described in Supporting Materials and Methods, which is published as supporting information on the PNAS web site. Experiment Overall Design: Three groups of 10 oocytes each were used. Reference RNA (100 Î¼g) was prepared by mixing 10 Î¼g of total RNA from each of 10 different normal human tissues, including skeletal muscle, kidney, lung, colon, liver, spleen, breast, brain, heart, and stomach (Ambion). Experiment Overall Design: Transcriptional profile of each sample was probed by using Affymetrix Human Genome U133 Plus 2.0 GeneChips.

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Epiblast production and thereby totipotency of the first two blastomeres is balanced or unbalanced depending on the region where the oocyte was fertilized

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