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.     

哺乳动物卵母细胞生发泡移植

生发泡(GV)移植是指将GV期卵母细胞的GV移入到去核的受体细胞(GV期卵母细胞、MII期卵母细胞或受精卵)透明带下,经融合形成一个重组卵的过程。GV移植对研究卵母细胞的细胞周期调控、成熟及受精时细胞核与细胞质之间的相互作用非常重要,可用于研究卵母细胞减数分裂异常和与年龄相关变化之间的关系及细胞质衰老与卵母细胞非整倍性之间的关系。现简要介绍了GV移植的基本程序,GV核体与胞质体的融合,重组卵的培养条件,重组卵成熟后的受精、人工激活和胚胎发育能力以及GV移植的意义。     

Large-scale chromatin remodeling in germinal vesicle bovine oocytes: interplay with gap junction functionality and developmental competence

In mammals, oocyte acquires a series of competencies sequentially during folliculogenesis that play critical roles at fertilization and early stages of embryonic development. In mouse, chromatin in germinal vesicle (GV) undergoes dynamic changes during oocyte growth and its progressive condensation has been related to the achievement of developmental potential. Cumulus cells are essential for the acquisition of meiotic competence and play a role in chromatin remodeling during oocyte growth. This study is aimed to characterize the chromatin configuration of growing and fully grown bovine oocytes, the status of communications between oocyte and cumulus cells and oocyte developmental potential. Following nuclear staining, we identified four discrete stages of GV, characterized by an increase of chromatin condensation. GV0 stage represented 82% of growing oocytes and it was absent in fully grown oocytes. GV1, GV2, and GV3 represented, respectively, 24, 31, and 45% of fully grown oocytes. Our data indicated a moderate but significant increase in oocyte diameter between GV0 and GV3 stage. By dye coupling assay the 98% of GV0 oocytes showed fully open communications while the number of oocytes with functionally closed communications with cumulus cells was significantly higher in GV3 group than GV1 and GV2. However, GV0 oocytes were unable to progress through metaphase II while GV2 and GV3 showed the highest developmental capability. We conclude that in bovine, the progressive chromatin condensation is related to the sequential achievement of meiotic and embryonic developmental competencies during oocyte growth and differentiation. Moreover, gap-junction-mediated communications between oocyte and cumulus cells could be implicated in modulating the chromatin remodeling process.     

Effect of maturation stage at cryopreservation on post-thaw cytoskeleton quality and fertilizability of equine oocytes

Oocyte cryopreservation is a potentially valuable technique for salvaging the germ-line when a valuable mare dies, but facilities for in vitro embryo production or oocyte transfer are not immediately available. This study examined the influence of maturation stage and freezing technique on the cryopreservability of equine oocytes. Cumulus oocyte complexes were frozen at the immature stage (GV) or after maturation in vitro for 30 hr (MII), using either conventional slow freezing (CF) or open pulled straw vitrification (OPS); cryoprotectant-exposed and untreated nonfrozen oocytes served as controls. After thawing, GV oocytes were matured in vitro, and MII oocytes were incubated for 0 or 6 hr, before staining to examine meiotic spindle quality by confocal microscopy. To assess fertilizability, CF MII oocytes were subjected to intracytoplasmic sperm injection (ICSI) and cultured in vitro. At 12, 24, and 48 hr after ICSI, injected oocytes were fixed to examine their progression through fertilization. Both maturation stage and freezing technique affected oocyte survival. The meiosis resumption rate was higher for OPS than CF for GV oocytes (28% vs. 1.2%; P < 0.05), but still much lower than for controls (66%). Cryopreserving oocytes at either stage induced meiotic spindle disruption (37%-67% normal spindles vs. 99% in controls; P < 0.05). Among frozen oocytes, however, spindle quality was best for oocytes frozen by CF at the MII stage and incubated for 6 hr post-thaw (67% normal); since this combination of cryopreservation/IVM yielded the highest proportion of oocytes reaching MII with a normal spindle (35% compared to <20% for other groups), it was used when examining the effects of cryopreservation on fertilizability. In this respect, the rate of normal fertilization for CF MII oocytes after ICSI was much lower than for controls (total oocyte activation rate, 26% vs. 56%; cleavage rate at 48 hr, 8% vs. 42%: P < 0.05). Thus, although IVM followed by CF yields a respectable percentage of normal-looking MII oocytes (35%), their ability to support fertilization is severely compromised.     

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.     

Combined use of dbcAMP and IBMX minimizes the damage induced by a long‐term artificial meiotic arrest in mouse germinal vesicle oocytes

Phosphodiesterase (PDE)‐mediated reduction of cyclic adenosine monophosphate (cAMP) activity can initiate germinal vesicle (GV) breakdown in mammalian oocytes. It is crucial to maintain oocytes at the GV stage for a long period to analyze meiotic resumption in vitro. Meiotic resumption can be reversibly inhibited in isolated oocytes by cAMP modulator forskolin, cAMP analog dibutyryl cAMP (dbcAMP), or PDE inhibitors, milrinone (Mil), Cilostazol (CLZ), and 3‐isobutyl‐1‐methylxanthine (IBMX). However, these chemicals negatively affect oocyte development and maturation when used independently. Here, we used ICR mice to develop a model that could maintain GV‐stage arrest with minimal toxic effects on subsequent oocyte and embryonic development. We identified optimal concentrations of forskolin, dbcAMP, Mil, CLZ, IBMX, and their combinations for inhibiting oocyte meiotic resumption. Adverse effects were assessed according to subsequent development potential, including meiotic resumption after washout, first polar body extrusion, early apoptosis, double‐strand DNA breaks, mitochondrial distribution, adenosine triphosphate levels, and embryonic development. Incubation with a combination of 50.0 μM dbcAMP and 10.0 μM IBMX efficiently inhibited meiotic resumption in GV‐stage oocytes, with low toxicity on subsequent oocyte maturation and embryonic development. This work proposes a novel method with reduced toxicity to effectively arrest and maintain mouse oocytes at the GV stage.     

Mouse-rabbit germinal vesicle transfer reveals that factors regulating oocyte meiotic progression are not species-specific in mammals

A series of experiments were designed to evaluate the meiotic competence of mouse oocyte germinal vesicle (GV) in rabbit ooplasm. In experiment 1, an isolated mouse GV was transferred into rabbit GV-stage cytoplast by electrofusion. It was shown that 71.8% and 63.3% of the reconstructed oocytes completed the first meiosis as indicated by the first polar body (PB1) emission when cultured in M199 and M199 + PMSG, respectively. Chromosomal analysis showed that 75% of matured oocytes contained the normal 20 mouse chromosomes. When mouse spermatozoa were microinjected into the cytoplasm of oocytes matured in M199 + PMSG and M199, as many as 59.4% and 48% finished the second meiosis as revealed by the second polar body (PB2) emission and a few fertilized eggs developed to the eight-cell stage. In experiment 2, a mouse GV was transferred into rabbit MII-stage cytoplast. Only 13.0-14.3% of the reconstructed oocytes underwent germinal vesicle breakdown (GVBD) and none proceeded past the MI stage. When two mouse GVs were transferred into an enucleated rabbit oocyte, only 8.7% went through GVBD. In experiment 3, a whole zona-free mouse GV oocyte was fused with a rabbit MII cytoplast. The GVBD rates were increased to 51.2% and 49.4% when cultured in M199 + PMSG and M199, respectively, but none reached the MII stage. In experiment 4, a mouse GV was transferred into a partial cytoplasm-removed rabbit MII oocyte in which the second meiotic apparatus was still present. GVBD occurred in nearly all the reconstructed oocytes when one or two GVs were transferred and two or three metaphase plates were observed in ooplasm after culturing in M199 + PMSG for 8 hr. These data suggest that cytoplasmic factors regulating the progression of the first and the second meioses are not species-specific in mammalian oocytes and that these factors are located in the meiotic apparatus and/or its surrounding cytoplasm at MII stage.     

Alterations and reversibility in the chromatin,cytoskeleton and development of pig oocytes treated with roscovitine

Germinal vesicle (GV) breakdown in mammalian oocytes is regulated by the activation of maturation promoting factor (MPF). We investigated a specific cdc2 kinase inhibitor, roscovitine, to maintain pig oocytes in the GV stage. Cumulus-oocyte complexes (COCs) were aspirated from slaughterhouse ovaries and cultured for 44 hr in NCSU#23 medium containing different levels of roscovitine (0, 10, 20, 30, 40, 50 microM in Experiment 1 and 0, 40, 60, 80, 100, 120 microM in Experiment 2). The COCs were cultured for another 44 hr after removal of the chemical. Twenty oocytes in each group were fixed at 44 hr for immunocytochemical labeling of the cytoskeleton and the rest (approximately 20/group) were fixed at the end of 88 hr after culture. Results showed that the inhibition of the oocyte in the GV stage was not effective when 10-50 microM (Experiment 1) of roscovitine were used (19-34%). When oocytes were released from the inhibitor, similar proportions (70-83%) of oocytes were observed in the MII or advanced stages among treatments. However, when higher concentrations of roscovitine were used (Experiment 2), significantly greater inhibitory effect was observed at the levels of 80-120 microM with 83-91% oocytes being blocked in the GV stage when compared to the control (9%) and the 40-60 microM (27-43%) groups (P < 0.05). Although 15-21% of the oocytes showed abnormal MII morphology with aberrant meiotic spindles and/or formation of cytoplasmic microtubules, a substantial number of oocytes resumed meiosis and reached MII stage at 44 hr after removal of this chemical. In Experiment 3, different concentrations of roscovitine (0, 20, 40, and 80 microM) were tested to examine the length of intervals (0, 11, 22, 33, and 44 hr) for an effective inhibition. Results showed that the inhibitory effect was significantly more prominent at 22 hr than that at 33 and 44 hr after roscovitine treatment in all treatment groups (P < 0.05). This study demonstrated that roscovitine-treated oocytes resumed meiosis after removal of the inhibitor. This could provide flexibility for studying porcine oocyte development and embryo cloning and may have application in other species.     

Position of the nucleus in mouse germinal vesicle–stage oocytes with different chromatin configurations

The mammalian germinal vesicle–stage (GV) oocytes are divided into two major types, NSN (non-surrounded nucleolus) and SN (surrounded nucleolus), and at least one intermediate type, pSN (partly surrounded nucleolus), based on large-scale chromatin configuration. In mice, the SN oocytes are considered to be the most meiotically competent, which explains active study of their phenotypic characteristics necessary for improvement of human reproductive technologies. One of such characteristics is the position of the GV (nucleus) relative to the center of the oocyte. However, the current data on this issue are contradictory and even completely absent for pSN oocytes. In this work, we have studied the GV position in 187 mouse GV oocytes belonging to NSN, SN, and pSN types using different approaches known from the literature. Our results suggest that (1) the most abundant in all examined types of oocytes are central GVs (43–66%) and the least abundant are peripheral GVs (12–39%); the pSN oocytes are closer to SN oocytes rather than to NSN oocytes according to the GV position; (3) the position of the nucleus in mouse GV oocytes is an ambiguous marker of large-scale chromatin configuration and, correspondingly, maturation competence of the oocyte; (4) the diversity of the GV position in NSN, SN, and pSN oocytes most likely reflects the ability of GVs to migrate; and (5) assessment of the GV position according to three variants (central, peripheral, and intermediate) is more informative as compared with two variants (central and peripheral).     

Effect of roscovitine on nuclear maturation, MPF and MAP kinase activity and embryo development of prepubertal goat oocytes

The low number of embryos obtained from IVM-IVF-IVC of prepubertal goat oocytes could be due to an incomplete cytoplasmic maturation. Roscovitine (ROS) inhibits MPF and MAP kinase activity and maintains the oocyte at Germinal Vesicle (GV) stage. The aim of this study was to determine if meiotic activity is arrested in prepubertal goat oocytes cultured with 0, 12.5, 25, 50 and 100 microM of ROS for 24 h. A group of oocytes from adult goats was cultured with 25 microM of ROS to compare the effect of ROS on prepubertal and adult goat oocytes. A sample of oocytes was stained to evaluate the nuclear stage at oocyte collection time and after ROS incubation. IVM-oocytes not exposed to ROS formed the control group. Prepubertal goat IVM-oocytes were inseminated and cultured for 8 days. The percentage of oocytes at GV stage, after exposition to ROS was significantly higher in adult goat oocytes (64.5%) than in prepubertal goat oocytes. No differences were found among 25, 50 and 100 microM ROS concentrations (29, 23 and 26%, oocytes at GV stage, respectively). After 8 days of culture, no differences in total embryos were observed between control oocytes and oocytes treated with 12.5 and 25 microM (45.2, 36.1 and 39.4%, respectively), however the percentage of blastocysts was higher in the control group. Western blot for the MAPK and p34(cdc2) showed that both enzymes were active in prepubertal goat oocytes after 24h of ROS exposition. In conclusion, a low percentage of prepubertal goat oocytes reached GV stage after ROS incubation; possibly because most of them had reinitiated the meiosis inside the follicle. ROS did not affect fertilization or total embryos but ROS showed a negative effect on blastocyst development.     

Chromosome condensation activity in the cytoplasm of anucleate and nucleate fragments of mouse oocytes

The activity of maturation promoting factor (MPF) which causes chromosome condensation and subsequent oocyte maturation was investigated in mouse oocytes using polyethylene-glycol-mediated cell fusion technique. Fully grown oocytes were bisected at germinal vesicle (GV) stage or shortly after germinal vesicle breakdown (GVBD) into anucleate and nucleate fragments. After 2-3 or 15-17 hr of culture these fragments were fused with interphase blastomeres from two-cell embryos. It was found that almost all the anucleate oocyte fragments cultured for a short term (2-3 hr), regardless of whether they were produced at GV stage or after GVBD, induced premature chromosome condensation in the blastomere nuclei, whereas only about 20% of those cultured for a long term (15-17 hr) could do so. On the other hand, the nucleate fragments always retain the cytoplasmic activity to induce chromosome condensation. Thus we suggested that the MPF initially could appear in mouse oocytes independently of the GV, that the mixing of GV material with the oocyte cytoplasm following GVBD had no effect on the activity of MPF in anucleate fragments, and that oocyte chromosomes or some components associated with them could play a significant role in maintaining the MPF activity.     

In vitro and in vivo studies reveal that hamster oocyte meiotic arrest is maintained only transiently by follicular fluid, but persistently by membrana/cumulus granulosa cell contact

Studies were carried out with the golden Syrian hamster to investigate the capacity of follicular fluid to maintain oocyte meiotic arrest and to determine the importance of cumulus-membrana granulosa cell contact in the regulation of meiotic status. The follicular fluid studies were conducted by cytological assessment of meiotic stage up to 6 hr after transferring cumulus-free oocytes into antra of explanted "host" follicles in vitro or into follicles of anesthetized animals prior to the gonadotropin surge at proestrus in vivo. The cumulus-membrana granulosa contact studies were undertaken with explanted follicles in which the oocyte-cumulus complex was dislodged from the underlying membrana granulosa, released into the antrum, and subsequently allowed to reestablish contact during 6 hr of incubation within the follicle. The extent of recontact of the dislodged complex with the underlying membrana granulosa was assessed visually at the end of incubation and was classified as close, moderate, or none. These various degrees of contact typically involved the following number of cumulus cells, as determined by serial sectioning of a representative sample of follicles after dislodgement and subsequent incubation: close, 32.7 +/- 1.78; moderate, 9.0 +/- 2.1; and no contact, 0. After 6 hr of incubation either in vitro or in vivo, few transferred oocytes remained at the germinal vesicle (GV) stage (18.8 +/- 8.7 and 17.3 +/- 4.0% GV, respectively). However, time course experiments revealed that meiotic resumption was significantly delayed in transferred oocytes compared with either liberated oocytes, spontaneously maturing oocytes, or follicle-enclosed oocytes induced to mature by luteinizing hormone in vitro (after 4 hr, transferred, 31.3 +/- 6.0% GV; liberated, 0% GV; follicle-enclosed, 0% GV; after 6 hr, 0% transferred oocytes exhibited a GV). In the dislodgement studies, after 6 hr of incubation, 26% of complexes reestablished close contact with the underlying membrana granulosa, 67% showed moderate contact, while 7% revealed no contact. There was a significant increase in the percentage GV stage oocytes as the extent of recontact increased (no contact, 21.9 +/- 3.6% GV; moderate contact, 56.6 +/- 6.8% GV; close contact, 87.5 +/- 14.4% GV). These data argue in favor of a stringent control of hamster oocyte meiotic status by the follicle cell/oocyte syncytium and against the possibility that follicular fluid is independently responsible for maintaining meiotic arrest.(ABSTRACT TRUNCATED AT 400 WORDS)     

Serial nuclear transfer improves the developmental potential of mouse embryos cloned from oocytes matured in a protein-free medium

Germinal vesicle (GV) oocytes matured in vitro are an alternative source for cytoplasmic recipients of nuclear transfer (NT). However, the developmental potential of oocytes matured in vitro is limited. In this study, we developed a protein-free maturation medium for mouse GV oocytes. Following parthenogenetic activation, the oocytes matured in the protein-free medium develop to blastocyst stage with a high efficiency, even up to the rate obtained from in vivo MII-oocytes (90.6% vs. 92.8%). Using the oocytes matured in the protein-free medium as the recipient, NT embryos develop to the blastocyst stage (17.6%). To further improve the developmental potential of NT embryos, we performed serial NT and compared the effect of three different activated cytoplasm samples derived from in vitro matured oocytes as the second recipient, that is, the effect of in vitro fertilized (IVF) zygote, the preactivated cytoplast and the IVF cytoplast, on the development of NT embryos. We found that when the pronucleus of NT zygote was transferred into the cytoplasm of the IVF zygote, the blastocyst formation increased to 39.4%. This is the first report to demonstrate the IVF zygote from oocytes matured in protein-free medium can be used successfully as the recipient for serial NT to enhance the developmental potential of mouse NT embryos from oocytes matured in the protein-free medium.