Oocyte Cytoplasmic Gas6 and Heparan Sulfate (HS) are Required to Establish the Open Chromatin State in Nuclei During Remodeling and Reprogramming

Abstract

Background/Aims: Previously, we found that silencing of growth arrest-specific gene 6 (Gas6) in oocytes impaired cytoplasmic maturation, resulting in failure of sperm chromatin decondensation (SCD) and pronuclear (PN) formation after fertilization. Thus, we conducted this study to determine the effect of Gas6 RNAi on downstream genes and to elucidate the working mechanism of Gas6 on oocyte cytoplasmic maturation and SCD. Methods: Using RT-PCR, Western blot and immunofluorescence, the expression levels of various target genes and the localization of heparan sulfate (HS) were analyzed after Gas6 RNAi. The roles of Gas6 in HS biosynthesis, production of ATP and GSH, ROS generation and ΔΨm were also investigated. SCD and micrococcal nuclease (MNase) analyses were used to examine the effects of HS on the open chromatin state in sperm and somatic cell nuclei, respectively. Results: Disruption of Gas6 expression led to the inhibition of HS biosynthesis through the reduction of several HS biosynthetic enzymes. The rescue experiment, HS treatment in vitro, significantly recovered SCD and PN formation, confirming that HS had the ability to induce sperm head remodeling during fertilization. Interestingly, excessive mitochondrial activation in Gas6-depleted MII oocytes caused ROS generation and glutathione (GSH) degradation via mitochondrial activation, such as elevated ΔΨm and ATP production. Indeed, HS-treated NIH3T3 cell nuclei showed an open chromatin state, as determined by diffuse DAPI staining and increased sensitivity to MNase. Conclusion: We propose that the addition of HS to sperm and/or oocyte maturation would improve the efficiency of in vitro fertilization and somatic cell nuclear transfer (SCNT) reprogramming.

Gas6 is a reciprocal regulator of mitophagy during mammalian oocyte maturation

“…Previously, we found by using JC-1 staining that Gas6 depletion induces mitochondrial overactivation and accumulation in the cytoplasm 8 . We hypothesized that the abnormal mitochondrial accumulation in response to decreased Gas6 may be due to the activation of PTPN11 and MTOR as well as reduction of BNIP3, known as an inactivator of MTOR 31 .…”

“…Previously, we reported that disruption of Gas6 resulted in insufficient oocyte cytoplasmic maturation via mitochondrial dysfunction such as mitochondrial overactivation and abnormal mitochondrial accumulation 8 . Based on the RNA-Seq analysis, we selected 2 genes related to mitochondrial functions ( Phyhipl and Tomm7 ) and 3 for mitophagy ( Atg2b , Binp3 and Esr2 ) and added Ptpn11 , as a downstream target of Gas6 (Fig.…”

“…However, depletion of Gas6 in oocytes results in failure of sperm chromatin remodeling and pronuclear formation through insufficient cytoplasmic maturation 7 . In a successive study, we found that the depletion of Gas6 in oocytes inhibited heparan sulfate biosynthesis and glutathione production via mitochondrial overactivation, characterized by increased ATP production and mitochondrial membrane potential 8 . Most importantly, Gas6 was a crucial factor for the completion of oocyte cytoplasmic maturation.…”

“…To date, two studies support Gas6 involvement in reproduction 7,8 . We first found that Gas6 is expressed in mouse oocytes and is not involved in the completion of the meiotic division since Gas6 -depleted oocytes mature to the metaphase II (MII) stage with extrusion of the first polar body 7 .…”

Corticosterone and testosterone treatment influence expression of gene pathways linked to meiotic segregation in preovulatory follicles of the domestic hen

“…Corticosterone treatment had the strongest effect, leading to the downregulation of several DEGs with the potential to adjust sex ratios based on 4 hypothesized mechanisms (Fig 5). EXT2 encodes a protein involved in the biosynthesis of heparin sulfate, a molecule located on the cell surface and known for its extreme negative charge due to sulfate and carboxyl groups [54,55]. Spindle function and movement of chromosomes within the cell is controlled by nanoscale electrostatic forces [56].…”