HDAC8 drives spindle organization during meiotic maturation of porcine oocytes

ObjectivesHistone deacetylase 8 (HDAC8) is one of the class I HDAC family proteins, which participates in the neuronal disorders, parasitic/viral infections, tumorigenesis and many other biological processes. However, its potential function during female germ cell development has not yet been fully understood.Materials and methodsHDAC8‐targeting siRNA was microinjected into GV oocytes to deplete HDAC8. PCI‐34051 was used to inhibit the enzyme activity of HDAC8. Immunostaining, immunoblotting and fluorescence intensity quantification were applied to assess the effects of HDAC8 depletion or inhibition on the oocyte meiotic maturation, spindle/chromosome structure, γ‐tubulin dynamics and acetylation level of α‐tubulin.ResultsWe observed that HDAC8 was localized in the nucleus at GV stage and then translocated to the spindle apparatus from GVBD to M II stages in porcine oocytes. Depletion of HDAC8 led to the oocyte meiotic failure by showing the reduced polar body extrusion rate. In addition, depletion of HDAC8 resulted in aberrant spindle morphologies and misaligned chromosomes due to the defective recruitment of γ‐tubulin to the spindle poles. Notably, these meiotic defects were photocopied by inhibition of HDAC8 activity using its specific inhibitor PCI‐34051. However, inhibition of HDAC8 did not affect microtubule stability as assessed by the acetylation level of α‐tubulin.ConclusionsCollectively, our findings demonstrate that HDAC8 acts as a regulator of spindle assembly during porcine oocyte meiotic maturation.     

Physiologically relevant miRNAs in mammalian oocytes are rare and highly abundant

miRNAs, ~22nt small RNAs associated with Argonaute (AGO) proteins, are important negative regulators of gene expression in mammalian cells. However, mammalian maternal miRNAs show negligible repressive activity and the miRNA pathway is dispensable for oocytes and maternal‐to‐zygotic transition. The stoichiometric hypothesis proposed that this is caused by dilution of maternal miRNAs during oocyte growth. As the dilution affects miRNAs but not mRNAs, it creates unfavorable miRNA:mRNA stoichiometry for efficient repression of cognate mRNAs. Here, we report that porcine ssc‐miR‐205 and bovine bta‐miR‐10b are exceptional miRNAs, which resist the diluting effect of oocyte growth and can efficiently suppress gene expression. Additional analysis of ssc‐miR‐205 shows that it has higher stability, reduces expression of endogenous targets, and contributes to the porcine oocyte‐to‐embryo transition. Consistent with the stoichiometric hypothesis, our results show that the endogenous miRNA pathway in mammalian oocytes is intact and that maternal miRNAs can efficiently suppress gene expression when a favorable miRNA:mRNA stoichiometry is established.     

Effect of age, GV transfer and modified nucleocytoplasmic ratio on PKCα in mouse oocytes and early embryos

Protein kinase C (PKC) is a family of Ser/Thr protein kinases that can be activated by Ca2+, phospholipid and diacylglycerol. There is evidence that PKC plays key roles in the meiotic maturation and activation of mammalian oocytes. The present study aimed to monitor the effect of age, germinal vesicle (GV) transfer and modified nucleoplasmic ratio on the subcellular distribution profile of PKCα, an important isozyme of PKC, in mouse oocytes undergoing meiotic maturation and following egg activation. Germinal vesicle oocytes were collected from 6-8-week-old and 12-month-old mice. Germinal vesicle-reconstructed oocytes and GV oocytes with one-half or one-third of the original oocyte volume were created using micromanipulation and electrofusion. The subcellular localization of PKCα was detected by immunocytochemistry and laser confocal microscopy. Our study showed that PKCα had a similar location pattern in oocytes and early embryos from young and old mice. PKCα was localized evenly in ooplasm, with weak staining in GV at the GV stage, and present in the entire meiosis II (MII) spindle at the MII stage. In pronuclear and 2-cell embryos, PKCα was concentrated in the nucleus except for the nucleolus. After the GV oocytes were reconstructed, the resultant MII oocytes and embryos showed a similar distribution of PKCα between reconstructed and unreconstructed controls. After one-half or two-thirds of the cytoplasm was removed from the GV oocytes, PKCα still had a similar location pattern in MII oocytes and early embryos from the GV oocytes with modified nucleoplasmic ratio. Our study showed that age, GV transfer and modified nucleocytoplasmic ratio does not affect distribution of PKCα during mouse oocyte maturation, activation, and early embryonic mitosis.     

Supplementation of mitochondria from endometrial mesenchymal stem cells improves oocyte quality in aged mice

Maternal ageing is one of the major causes of reduced ovarian reserve and low oocyte quality in elderly women. Decreased oocyte quality is the main cause of age‐related infertility. Mitochondria are multifunctional energy stations that determine the oocyte quality. The mitochondria in aged oocytes display functional impairments with mtDNA damage, which leads to reduced competence and developmental potential of oocytes. To improve oocyte quality, mitochondrial supplementation is carried out as a potential therapeutic approach. However, the selection of suitable cells as the source of mitochondria remains controversial. We cultivated endometrial mesenchymal stem cells (EnMSCs) from aged mice and extracted mitochondria from EnMSCs. To improve the quality of oocytes, GV oocytes were supplemented with mitochondria via microinjection. And MII oocytes from aged mice were fertilized by intracytoplasmic sperm injection (ICSI), combining EnMSCs'' mitochondrial microinjection. In this study, we found that the mitochondria derived from EnMSCs could significantly improve the quality of aged oocytes. Supplementation with EnMSC mitochondria significantly increased the blastocyst ratio of MII oocytes from aged mice after ICSI. We also found that the birth rate of mitochondria‐injected ageing oocytes was significantly increased after embryo transplantation. Our study demonstrates that supplementation with EnMSC‐derived mitochondria can improve the quality of oocytes and promote embryo development in ageing mice, which might provide a prospective strategy for clinical treatment.

In this study, we chose endometrial mesenchymal stem cells (EnMSCs) as the sources of mitochondria. We isolated the EnMSCs from 10‐month‐old mice and then extracted the mitochondria of EnMSCs. Then, the GV oocytes and MII oocytes from aged mice were injected with mitochondria. We found that mitochondria derived from EnMSCs could significantly improve the quality of oocytes, promote the embryonic development and improve the birth rates of aged mice.     

Cytoskeleton Genes Expression and Survival Rate Comparison Between Immature and Mature Yak Oocyte After OPS Vitrification

This study was conducted to investigate the effect of vitrification on survival rate and cytoskeleton gene expression during yak oocyte maturation. The yak oocytes were incubated for 0?h [germinal vesicle (GV) stage] and in vitro matured for 24?h [metaphase II (MII) stage] to obtain immature and mature oocytes. Survival rate after vitrification were compared between immature and mature yak oocytes and cytoskeleton-related genes [cytokeratin 8 (CK8), β-actin (ACTB), and gap junction protein, alpha 1 (GJA1)] were tested by real-time PCR. Our results showed that MII stage survival rate after open pulled straw vitrification (35.60%) is significantly higher than GV stage (25.90%) oocytes. Furthermore, expression of CK8, ACTB, and GJA1 in MII stage oocytes are also significantly higher than GV stage oocytes. In conclusion, our study demonstrated that higher expression of GJA1, CK8, and ACTB in vitrify-warmed MII stage oocytes when compared with GV stage oocytes and such discrepancy might result in higher survival rate in vitrify-warmed MII stage oocytes.     

Production of non-mosaic genome edited porcine embryos by injection of CRISPR/Cas9 into germinal vesicle oocytes

Genetically modified pigs represent a great promise for generating models of human diseases and producing new breeds.Generation of genetically edited pigs using somatic cell nuclear transfer(SCNT)or zygote cytoplasmic microinjection is a tedious process due to the low developmental rate or mosaicism of the founder(FO).Herein,we developed a method termed germinal vesicle oocyte gene editing(GVGE)to produce non-mosaic porcine embryos by editing maternal alleles during the GV to MII transition.Injection of Cas9 mRNA and X-linked Dmd gene-specific gRNA into GV oocytes did not affect their developmental potential.The MII oocytes edited during in vitro maturation(IVM)could develop into blastocysts after parthenogenetic activation(PA)or in vitro fertilization(IVF).Genotyping results indicated that the maternal gene X-linked Dmd could be efficiently edited during oocyte maturation.Up to81.3% of the edited IVF embryos were non-mosaic Dmd gene mutant embryos.In conclusion,GVGE might be a valuable method for the generation of non-mosaic maternal allele edited FO embryos in a short simple step.     

The G protein coupled receptor 3 is involved in cAMP and cGMP signaling and maintenance of meiotic arrest in porcine oocytes

The arrest of meiotic prophase in mammalian oocytes within fully grown follicles is dependent on cyclic adenosine monophosphate (cAMP) regulation. A large part of cAMP is produced by the Gs-linked G-protein-coupled receptor (GPR) pathway. In the present study, we examined whether GPR3 is involved in the maintenance of meiotic arrest in porcine oocytes. Expression and distribution of GPR3 were examined by western blot and immunofluorescence microscopy, respectively. The results showed that GPR3 was expressed at various stages during porcine oocyte maturation. At the germinal vesicle (GV) stage, GPR3 displayed a maximal expression level, and its expression remained stable from pro-metaphase I (MI) to metaphase II (MII). Immunofluorescence staining showed that GPR3 was mainly distributed at the nuclear envelope during the GV stage and localized to the plasma membrane at pro-MI, MI and MII stages. RNA interference (RNAi) was used to knock down the GPR3 expression within oocytes. Injection of small interfering double-stranded RNA (siRNA) targeting GPR3 stimulated meiotic resumption of oocytes. On the other hand, overexpression of GPR3 inhibited meiotic maturation of porcine oocytes, which was caused by increase of cGMP and cAMP levels and inhibition of cyclin B accumulation. Furthermore, incubation of porcine oocytes with the GPR3 ligand sphingosylphosphorylcholine (SPC) inhibited oocyte maturation. We propose that GPR3 is required for maintenance of meiotic arrest in porcine oocytes through pathways involved in the regulation of cAMP and cGMP.     

NEK5 regulates cell cycle progression during mouse oocyte maturation and preimplantation embryonic development

NEK5, a member of never in mitosis‐gene A‐related protein kinase, is involved in the regulation of centrosome integrity and centrosome cohesion at mitosis in somatic cells. In this study, we investigated the expression and function of NEK5 during mouse oocyte maturation and preimplantation embryonic development. The results showed that NEK5 was expressed from germinal vesicle (GV) to metaphase II (MII) stages during oocyte maturation with the highest level of expression at the GV stage. It was shown that NEK5 localized in the cytoplasm of oocytes at GV stage, concentrated around chromosomes at germinal vesicle breakdown (GVBD) stage, and localized to the entire spindle at prometaphase I, MI and MII stages. The small interfering RNA‐mediated depletion of Nek5 significantly increased the phosphorylation level of cyclin‐dependent kinase 1 in oocytes, resulting in a decrease of maturation‐promoting factor activity, and severely impaired GVBD. The failure of meiotic resumption caused by Nek5 depletion could be rescued by the depletion of Wee1B. We found that Nek5 depletion did not affect CDC25B translocation into the GV. We also found that NEK5 was expressed from 1‐cell to blastocyst stages with the highest expression at the blastocyst stage, and Nek5 depletion severely impaired preimplantation embryonic development. This study demonstrated for the first time that NEK5 plays important roles during meiotic G2/M transition and preimplantation embryonic development.     

Epigenetic status of the H19 locus in human oocytes following in vitro maturation

Imprinting is an epigenetic modification that is reprogrammed in the germ line and leads to the monoallelic expression of some genes. Imprinting involves DNA methylation. Maternal imprint is reset during oocyte growth and maturation. In vitro maturation (IVM) of oocytes may, therefore, interfere with imprint acquisition and/or maintenance. To evaluate if maturing human oocytes in vitro would be hazardous at the epigenetic level, we first determined the methylation profile of the H19 differentially methylated region (DMR). The methylation status of the H19 DMR seems particularly vulnerable to in vitro culture conditions. We analyzed oocytes at different stages of maturation following IVM, germinal vesicle (GV), metaphase I (MI), and metaphase II (MII), using the bisulfite mutagenesis technique. Our results indicated that the unmethylated specific maternal profile for the H19 DMR was stably established at the GV stage. The majority of MI-arrested oocytes exhibited an altered pattern of methylation, the CTCF-binding site being methylated in half of the DNA strands analyzed. Of the 20 MII oocytes analyzed, 15 showed the normal unmethylated maternal pattern, while 5 originating from two different patients exhibited a methylated pattern. These findings highlight the need for extended analysis on MII-rescued oocytes to appreciate the epigenetic safety of the IVM procedure, before it becomes a routine and practical assisted reproductive procedure.