The RNA ligase RNA terminal phosphate cyclase B regulates mRNA alternative splicing and is required for mouse oocyte development and maintenance.

Recent large-scale mRNA sequencing has shown that introns are retained in 5-10% of mRNA, and these events are named intron retention (IR). IR has been recognized as a key mechanism in the regulation of gene expression. However, the role of this mechanism in female reproduction in mammals remains unclear. RNA terminal phosphate cyclase B (RTCB) is a RNA ligase; we found that RTCB conditional knockout mice have premature ovarian failure and that RTCB plays a crucial role in follicular development. RTCB regulated the splicing of transcripts related to DNA methylation and DNA damage repair. In addition, it regulated the resumption of oocyte meiosis by affecting CDK1 activation. Moreover, the loss of RTCB suppressed zygotic genome activation (ZGA) and decreased translation at the global level. In addition, Rtcb deletion resulted in the accumulation of maternal mRNAs containing unspliced introns and in a decline in the overall level of transcripts. As a result, the Rtcb-/- females were sterile. Our study highlights the important role of RTCB-regulated noncanonical alternative splicing in female reproduction.

Potential role for human menopausal gonadotropin in ovine superovulatory regimens during the breeding season.

Human menopausal gonadotrophin (hMG) has been reported to produce a comparable superovulatory response to that of follicle stimulating hormone (FSH). Furthermore, hMG has a long half-life as compared with FSH. The present study was designed to compare hMG administered once daily and FSH administered twice daily over a 4 - day period on superovulatory response of Suffolk ewes. During the mid-luteal phase, twenty-four Suffolk donor ewes received intravaginal sponges at day 0 for 12 days. The superovulatory regimens in the Control group (n = 12) and the Treatment group (n = 12) consisted of eight injections of FSH given at twice daily and four injections of hMG given at once daily, respectively. At day 13, the donor ewes were subjected to laparoscopic insemination. Embryos were recovered, classified, and transferred to recipient ewes at day 19. Pregnancy status was determined by ultrasound examination 40 days after transfer. Lambing rate was calculated after all the ewes had delivered. No significant differences were observed between the two groups in terms of the structures recovered, transferable embryos, degenerated embryos, unfertilized oocytes, pregnancy rate and lambing rate. The results showed that once daily injection of hMG can produce a comparable superovulatory response and embryo transfer outcomes to those obtained by twice daily injection of FSH over a 4 - day period. It is feasible that hMG is used to replace FSH and reduce the number of injection treatments in ovine superovulatory regimens.

Prolonging the time of semen deposition increases the pregnancy rates of ewes subjected to fixed time cervical insemination during the breeding season.

The main limiting factor of artificial cervical insemination in ewes is the long and narrow fibrous cervical canal, which impedes the transport of spermatozoa and leads to lower pregnancy rates. The hypothesis that prolonging the time of semen deposition during ovine cervical insemination can increase pregnancy rates was investigated in this study. Estrus was synchronized in 150 multiparous Ujimqin ewes using a polyurethane intravaginal sponge impregnated with 45 mg of flurogestone acetate. The sponge was left in the vagina for 12 days followed by an injection of 330 IU of eCG at sponge removal. After the exclusion of two ewes due to sponge loss, the remaining 148 ewes were divided into the Treatment group (n = 75) and the Control group (n = 73). Each ewe was inseminated once between 56 and 60 h after the removal of sponges, using a new type of insemination device containing 0.25 ml of diluted semen. Semen was collected from eight Black Suffolk rams and all the ejaculates were pooled and diluted in ultra-high temperature-treated commercial skimmed milk. The time of semen deposition was prolonged to 60 s in the Treatment group, while ewes were given traditional insemination in the Control group. Pregnancy status was determined by transabdominal ultrasound examination 45 days after insemination. Lambing performance was calculated after all the ewes had been delivered. Significant differences were observed between the Treatment group and the Control group in terms of the pregnancy rate and the fecundity rate (73.3% and 93.3% vs 56.2% and 71.2%, p < .05 and p < .01, respectively). In conclusion, prolonging the time of semen deposition significantly increased pregnancy and fecundity rates in estrus-synchronized Ujimqin ewes subjected to fixed time cervical insemination.

Rescue of failed oocyte activation after ICSI in a mouse model of male factor infertility by recombinant phospholipase Cζ.

Artificial oocyte activation to overcome failed fertilization after intracytoplasmic sperm injection (ICSI) in human oocytes typically employs Ca(2+) ionophores to produce a single cytosolic Ca(2+) increase. In contrast, recombinant phospholipase Czeta (PLCζ) causes Ca(2+) oscillations indistinguishable from those occurring during fertilization, but remains untested for its efficacy in a scenario of ICSI fertilization failure. Here, we compare PLCζ with other activation stimuli in a mouse model of failed oocyte activation after ICSI, in which heat-treated sperm are injected into mouse oocytes. We show that increasing periods of 56 °C exposure of sperm produces a progressive loss of Ca(2+) oscillations after ICSI. The decrease in Ca(2+) oscillations produces a reduction in oocyte activation and embryo development to the blastocyst stage. We treated such oocytes that failed to activate after ICSI either with Ca(2+) ionophore, or with Sr(2+) media which causes Ca(2+) oscillations, or we injected them with recombinant human PLCζ. All these treatments rescued oocyte activation, although Sr(2+) and PLCζ gave the highest rates of development to blastocyst. When recombinant PLCζ was given to oocytes previously injected with control sperm, they developed normally to the blastocyst stage at rates similar to that after control ICSI. The data suggest that recombinant human PLCζ protein is an efficient means of rescuing oocyte activation after ICSI failure and that it can be effectively used even if the sperm already contains endogenous Ca(2+) releasing activity.

Zygotic Splicing Activation of the Transcriptome is a Crucial Aspect of Maternal-to-Zygotic Transition and Required for the Conversion from Totipotency to Pluripotency.

During maternal-to-zygotic transition (MZT) in the embryo, mRNA undergoes complex post-transcriptional regulatory processes. However, it is unclear whether and how alternative splicing plays a functional role in MZT. By analyzing transcriptome changes in mouse and human early embryos, dynamic changes in alternative splicing during MZT are observed and a previously unnoticed process of zygotic splicing activation (ZSA) following embryonic transcriptional activation is described. As the underlying mechanism of RNA splicing, splicing factors undergo dramatic maternal-to-zygotic conversion. This conversion relies on the key maternal factors BTG4 and PABPN1L and is zygotic-transcription-dependent. CDK11-dependent phosphorylation of the key splicing factor, SF3B1, and its aggregation with SRSF2 in the subnuclear domains of 2-cell embryos are prerequisites for ZSA. Isoforms generated by erroneous splicing, such as full-length Dppa4, hinder normal embryonic development. Moreover, alternative splicing regulates the conversion of early embryonic blastomeres from totipotency to pluripotency, thereby affecting embryonic lineage differentiation. ZSA is an essential post-transcriptional process of MZT and has physiological significance in generating new life. In addition to transcriptional activation, appropriate expression of transcript isoforms is also necessary for preimplantation embryonic development.

Improved Cell Properties of Human Dental Pulp Stem Cells (hDPSCs) Isolated and Expanded in a GMP Compliant and Xenogeneic Serum-free Medium.

BACKGROUND/AIM: Human dental pulp mesenchymal stem cells (hDPSCs) are considered to be a good cell source for cell-based clinical therapy, due to the advantages of high proliferation capacity, multilineage differentiation potential, immune regulation abilities, less ethnic concerns and non-invasive access. However, hDPSCs were traditionally isolated and expanded in medium containing fetal bovine serum (FBS), which is a barrier for clinical application due to the safety issues (virus transmission and allergy). Although many studies make efforts to screen out a suitable culture medium, the results are not promising so far. Therefore, a standard good manufacturing practice (GMP) compliant culture system is urgently required for the large-scale cell production. This study aimed to find suitable culture conditions for producing clinical grade hDPSCs to meet the requirements for clinical cell-based therapy and further to promote the application of hDPSCs into tissue regeneration or disease cure. MATERIALS AND METHODS: We derived hDPSCs from nine orthodontic teeth expanded in two different media: a GMP compliant and xenogeneic serum-free medium (AMMS) and a serum containing medium (SCM). Cell propterties including morphology, proliferation, marker expression, differentiation, stemness, senescence and cytokine secretion between these two media were systematically compared. RESULTS: hDPSCs cultured in both media exhibited the typical characteristics of mesenchymal stem cells (MSCs). However, we found that more cell colonies formed in the primary culture in AMMS, and the hDPSCs displayed higher proliferation capacity, differentiation potential and better stemness maintenance during sub-culturing in AMMS. CONCLUSION: Cell properties of hDPSCs could be improved when they were isolated and expanded in AMMS, which might provide a good candidate of culture medium for large-scale cell manufacturing.

Divergent effect of mammalian PLCζ in generating Ca²âº oscillations in somatic cells compared with eggs.

Sperm PLCζ (phospholipase Cζ) is a distinct phosphoinositide-specific PLC isoform that is proposed to be the physiological trigger of egg activation and embryo development at mammalian fertilization. Recombinant PLCζ has the ability to trigger Ca²âº oscillations when expressed in eggs, but it is not known how PLCζ activity is regulated in sperm or eggs. In the present study, we have transfected CHO (Chinese-hamster ovary) cells with PLCζ fused with either YFP (yellow fluorescent protein) or luciferase and found that PLCζ-transfected cells did not display cytoplasmic Ca²âº oscillations any differently from control cells. PLCζ expression was not associated with changes in CHO cell resting Ca²âº levels, nor with a significantly changed Ca²âº response to extracellular ATP compared with control cells transfected with either YFP alone, a catalytically inactive PLCζ or luciferase alone. Sperm extracts containing PLCζ also failed to cause Ca²âº oscillations in CHO cells. Despite these findings, PLCζ-transfected CHO cell extracts exhibited high recombinant protein expression and PLC activity. Furthermore, either PLCζ-transfected CHO cells or derived cell extracts could specifically cause cytoplasmic Ca²âº oscillations when microinjected into mouse eggs. These data suggest that PLCζ-mediated Ca²âº oscillations may require specific factors that are only present within the egg cytoplasm or be inhibited by factors present only in somatic cell lines.

Different occurrence rates of centrally located cytoplasmic granulation in one cohort oocytes show distinctive embryo competence and clinical outcomes.

Centrally located cytoplasmic granulation (central granulation) is a common cytoplasmic dysmorphism in human oocytes retrieved after controlled ovarian hyperstimulation (COH). In order to achieve a better understanding of its formation and effects on clinical outcomes, we retrospectively analyzed 422 ICSI treatment cycles. Three groups of patients were classified according to the ratio of central granulation occurrence in one egg cohort, as partial granulation, all granulation and control groups. The partial granulation group had a significantly lower BMI and higher AMH level compared to the control or all granulation groups. Consistent with these distinctive features in the partial granulation group, fertilization and blastocyst formation rates were reduced significantly in the partial granulation group but not in the all granulation group. Furthermore, the clinical outcomes in fresh embryo transfer cycles were dramatically reduced in the partial granulation group compared with the control group. However, in FET cycles, all three clinical outcomes were significantly reduced in the all granulation group but not in the partial granulation group. We propose that partial granulation may reflect a specific population of patients, and that the central granulation structure is sensitive to cryopreservation.

Redistribution of mitochondria leads to bursts of ATP production during spontaneous mouse oocyte maturation.

During mammalian oocyte maturation there are marked changes in the distribution of mitochondria that supply the majority of the cellular ATP. Such redistribution of mitochondria is critical for oocyte quality, as oocytes with a poor developmental potential display aberrant mitochondrial distribution and lower ATP levels. Here we have investigated the dynamics of mitochondrial ATP production throughout spontaneous mouse oocyte maturation, using live measurements of cytosolic and mitochondrial ATP levels. We have observed three distinct increases in cytosolic ATP levels temporally associated with discrete events of oocyte maturation. These changes in cytosolic ATP levels are mirrored by changes in mitochondrial ATP levels, suggesting that mitochondrial ATP production is stimulated during oocyte maturation. Strikingly, these changes in ATP levels correlate with the distribution of mitochondria undergoing translocation to the peri-nuclear region and aggregation into clusters. Mitochondrial clustering during oocyte maturation was concomitant with the formation of long cortical microfilaments and could be disrupted by cytochalasin B treatment. Furthermore, the ATP production bursts observed during oocyte maturation were also inhibited by cytochalasin B suggesting that mitochondrial ATP production is stimulated during oocyte maturation by microfilament-driven, sub-cellular targeting of mitochondria.

Use of luciferase chimaera to monitor PLCzeta expression in mouse eggs.

The microinjection of cRNA encoding phospholipase Czeta (PLC zeta) causes Ca2+ oscillations and the activation of development in mouse eggs. The PLCzeta protein that is expressed in eggs after injection of cRNA is effective in causing Ca2+ oscillations at very low concentrations. In order to measure the amount and timecourse of protein expression we have tagged PLCzeta with firefly luciferase. The expression of the luciferase protein tag in eggs is then measured by incubation in luciferin combined with luminescence imaging, or by the lysis of eggs in the presence of Mg-ATP and luciferin in a luminometer. The use of luciferase to monitor protein expression after injection of cRNA is a sensitive and effective method that efficiently allows for sets of eggs to be used for PLCzeta quantitation, Ca2+ imaging, and studies of embryo development.

Regulation of diacylglycerol production and protein kinase C stimulation during sperm- and PLCzeta-mediated mouse egg activation.

BACKGROUND INFORMATION: At fertilization in mammalian eggs, the sperm induces a series of Ca(2+) oscillations via the production of inositol 1,4,5-trisphosphate. Increased inositol 1,4,5-trisphosphate production appears to be triggered by a sperm-derived PLCzeta (phospholipase C-zeta) that enters the egg after gamete fusion. The specific phosphatidylinositol 4,5-bisphosphate hydrolytic activity of PLCzeta implies that DAG (diacylglycerol) production, and hence PKC (protein kinase C) stimulation, also occurs during mammalian egg fertilization. Fertilization-mediated increase in PKC activity has been demonstrated; however, its precise role is unclear. RESULTS: We investigated PLCzeta- and fertilization-mediated generation of DAG in mouse eggs by monitoring plasma-membrane translocation of a fluorescent DAG-specific reporter. Consistent plasma-membrane DAG formation at fertilization, or after injection of physiological concentrations of PLCzeta, was barely detectable. However, when PLCzeta is overexpressed in eggs, significant plasma-membrane DAG production occurs in concert with a series of unexpected secondary high-frequency Ca(2+) oscillations. We show that these secondary Ca(2+) oscillations can be mimicked in a variety of situations by the stimulation of PKC and that they can be prevented by PKC inhibition. The way PKC leads to secondary Ca(2+) oscillations appears to involve Ca(2+) influx and the loading of thapsigargin-sensitive Ca(2+) stores. CONCLUSIONS: Our results suggest that overproduction of DAG in PLCzeta-injected eggs can lead to PKC-mediated Ca(2+) influx and subsequent overloading of Ca(2+) stores. These results suggest that DAG generation in the plasma membrane of fertilizing mouse eggs is minimized since it can perturb egg Ca(2+) homoeostasis via excessive Ca(2+) influx.

The dynamics of calcium oscillations that activate mammalian eggs.

It has been known for some time that mammalian eggs are activated by a series of intracellular calcium oscillations that occur shortly after sperm egg membrane fusion. Recent work has identified a novel sperm specific phospholipase C zeta as the likely agent that stimulates the calcium oscillations in eggs after sperm-egg membrane fusion. PLCzeta is stimulated by low intracellular calcium levels in a manner which suggests that there is a regenerative feedback of calcium release and PLCzeta induced inositol 1,4,5-trisphophate (InsP3) production in eggs. This implies calcium oscillations in fertilizing mammalian eggs are driven by underlying oscillations of InsP3. This model of oscillations is supported by the response of mouse eggs to sudden increases in InsP3. The cellular targets of calcium oscillations include calmodulin-dependent protein kinases, protein kinase C and mitochondria. There is evidence that eggs might be best activated by multiple calcium increases rather than a single calcium rise. As yet we do not fully understand how the target of calcium in a mammalian egg might decode the patterns of calcium changes that can occur during egg activation.

The effect of follicle-stimulating hormone on follicular development, granulosa cell apoptosis and steroidogenesis and its mediation by insulin-like growth factor-I in the goat ovary.

The effect of FSH on goat follicular development, granulosa cell apoptosis and steroidogenesis and its mediation by insulin-like growth factor (IGF)-I were studied through both in vivo and in vitro experiments. The FSH treatment was begun on Day 9 after estrus and consisted of injections twice a day for 3 days in decreasing doses (7.5-7.5-5.0-5.0-2.5-2.5 mg). Does in both treatment and control groups were slaughtered for ovaries on Day 12. Granulosa cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Expression of IGF-I and IGF-II mRNA was determined by RT-PCR, while concentrations of progesterone (P4), estradiol (E2), IGF-I and IGF-II were measured by radioimmunoassay (RIA). Following parameters increased significantly (P<0.05) after the FSH treatment: follicle number (5.0+/-1.5 versus 9.0+/-2.0 per ovary), the level of E2 (0.1+/-0.1 ng/ml versus 0.7+/-0.2 ng/ml), the E2/P4 ratio (0.7+/-0.4 versus 4.7+/-3.0) and the concentrations of IGF-I (0.5+/-0.2 ng/ml versus 119.4+/-15.1 ng/ml) and IGF-II (0.12+/-0.03 ng/ml versus 40.9+/-18.7 ng/ml) in follicular fluid of the medium sized (3-5 mm) follicles and in the ovarian cortex the relative quantity of IGF-I mRNA (0.37+/-0.17 versus 0.90+/-0.12 Max OD). In contrast, the ratio of apoptotic granulosa cells in these follicles was reduced significantly (0.53+/-0.1 versus 0.10+/-0.01, P<0.05). In large (>5 mm) follicles, however, only the follicle number (2.3+/-0.7 versus 7.0+/-1.5 per ovary) and the level of IGF-I (38.4+/-11.0 ng/ml versus 87.3+/-13.9 ng/ml) increased significantly (P<0.05), whereas other values did not change. In vitro culture of granulosa cells showed that FSH significantly (P<0.05) enhanced IGF-I production (12.7+/-2.1 ng/ml versus 26.+/-21.9 ng/ml) by these cells, and both FSH and IGF-I reduced the ratios of apoptotic cells (from 0.7+/-0.07 to 0.3+/-0.1 and 0.2+/-0.04, respectively) and the effect was additive when both were used together. H89, the PKA pathway inhibitor, blocked the effect of FSH on granulosa cell apoptosis and IGF-I production in vitro. These results indicated that FSH mainly enhanced the development of medium sized follicles in the goat by suppressing the apoptosis of granulosa cells via increasing production of IGF-I and steroids, possibly through the PKA pathway.

Phospholipase Cζ rescues failed oocyte activation in a prototype of male factor infertility.

OBJECTIVE: To determine the effect of infertility-linked sperm phospholipase Cζ (PLCζ) mutations on their ability to trigger oocyte Ca(2+) oscillations and development, and also to evaluate the potential therapeutic utility of wild-type, recombinant PLCζ protein for rescuing failed oocyte activation and embryo development. DESIGN: Test of a novel therapeutic approach to male factor infertility. SETTING: University medical school research laboratory. PATIENT(S): Donated unfertilized human oocytes from follicle reduction. INTERVENTION(S): Microinjection of oocytes with recombinant human PLCζ protein or PLCζ cRNA and a Ca(2+)-sensitive fluorescent dye. MAIN OUTCOME MEASURE(S): Measurement of the efficacy of mutant and wild-type PLCζ-mediated enzyme activity, oocyte Ca(2+) oscillations, activation, and early embryo development. RESULT(S): In contrast to the wild-type protein, mutant forms of human sperm PLCζ display aberrant enzyme activity and a total failure to activate unfertilized oocytes. Subsequent microinjection of recombinant human PLCζ protein reliably triggers the characteristic pattern of cytoplasmic Ca(2+) oscillations at fertilization, which are required for normal oocyte activation and successful embryo development to the blastocyst stage. CONCLUSION(S): Dysfunctional sperm PLCζ cannot trigger oocyte activation and results in male factor infertility, so a potential therapeutic approach is oocyte microinjection of active, wild-type PLCζ protein. We have demonstrated that recombinant human PLCζ can phenotypically rescue failed activation in oocytes that express dysfunctional PLCζ, and that this intervention culminates in efficient blastocyst formation.

PLCζ causes Ca(2+) oscillations in mouse eggs by targeting intracellular and not plasma membrane PI(4,5)P(2).

Sperm-specific phospholipase C ζ (PLCζ) activates embryo development by triggering intracellular Ca(2+) oscillations in mammalian eggs indistinguishable from those at fertilization. Somatic PLC isozymes generate inositol 1,4,5-trisphophate-mediated Ca(2+) release by hydrolyzing phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) in the plasma membrane. Here we examine the subcellular source of PI(4,5)P(2) targeted by sperm PLCζ in mouse eggs. By monitoring egg plasma membrane PI(4,5)P(2) with a green fluorescent protein-tagged PH domain, we show that PLCζ effects minimal loss of PI(4,5)P(2) from the oolemma in contrast to control PLCδ1, despite the much higher potency of PLCζ in eliciting Ca(2+) oscillations. Specific depletion of this PI(4,5)P(2) pool by plasma membrane targeting of an inositol polyphosphate-5-phosphatase (Inp54p) blocked PLCδ1-mediated Ca(2+) oscillations but not those stimulated by PLCζ or sperm. Immunolocalization of PI(4,5)P(2), PLCζ, and catalytically inactive PLCζ (ciPLCζ) revealed their colocalization to distinct vesicular structures inside the egg cortex. These vesicles displayed decreased PI(4,5)P(2) after PLCζ injection. Targeted depletion of vesicular PI(4,5)P(2) by expression of ciPLCζ-fused Inp54p inhibited the Ca(2+) oscillations triggered by PLCζ or sperm but failed to affect those mediated by PLCδ1. In contrast to somatic PLCs, our data indicate that sperm PLCζ induces Ca(2+) mobilization by hydrolyzing internal PI(4,5)P(2) stores, suggesting that the mechanism of mammalian fertilization comprises a novel phosphoinositide signaling pathway.

Rhythmic actomyosin-driven contractions induced by sperm entry predict mammalian embryo viability.

Fertilization-induced cytoplasmic flows are a conserved feature of eggs in many species. However, until now the importance of cytoplasmic flows for the development of mammalian embryos has been unknown. Here, by combining a rapid imaging of the freshly fertilized mouse egg with advanced image analysis based on particle image velocimetry, we show that fertilization induces rhythmical cytoplasmic movements that coincide with pulsations of the protrusion forming above the sperm head. We find that these movements are caused by contractions of the actomyosin cytoskeleton triggered by Ca(2+) oscillations induced by fertilization. Most importantly, the relationship between the movements and the events of egg activation makes it possible to use the movements alone to predict developmental potential of the zygote. In conclusion, this method offers, thus far, the earliest and fastest, non-invasive way to predict the viability of eggs fertilized in vitro and therefore can potentially improve greatly the prospects for IVF treatment.

Requirements of Src family kinase during meiotic maturation in mouse oocyte.

The Src family kinase (SFK) is important in normal cell cycle control. However, its role in meiotic maturation in mammalian has not been examined. We used confocal microscope immunofluorescence to examine the in vitro dynamics of the subcellular distribution of SFK during the mouse oocyte meiotic maturation and further evaluated the functions of SFK via biochemical analysis using a specific SFK pharmacological inhibitor, PP(2). Our results showed that nonphospho-SFK was absent in oocyte upon its release from follicle. Nonphospho-SFK appeared in cytoplasm 0.5 hr after the release of oocyte and translocated to germinal vesicle (GV) before germinal vesicle breakdown (GVBD). After GVBD, nonphospho-SFK colocated with condensed chromosomes. In occyte at metaphase I (MI) and telophase I, nonphospho-SFK accumulated in the cortex and the cleavage furrow respectively besides its existence in cytoplasm in both stages. In oocyte at metaphase II (MII), nonphospho-SFK concentrated at the aligned chromosomes. In contrast, phospho-SFK was absent in oocyte until 1 hr after its release from the follicle. Phospho-SFK accumulated in the GV, the cortex, and cytoplasm immediately prior to GVBD. After GVBD, phospho-SFK evenly distributed in oocyte. In oocyte at MII, phospho-SFK localized throughout the cytoplasm and under the egg member. When the SFK activity was inhibited, the oocyte failed to initiate GVBD, could not go into MII, and could not extrude the first polar body. Our results demonstrated that SFK is required for meiotic maturation in mouse oocyte.

The genetic heterozygosity and fitness of tetraploid embryos and embryonic stem cells are crucial parameters influencing survival of mice derived from embryonic stem cells by tetraploid embryo aggregation.

The aim of this paper was to determine whether the genetic background of tetraploid embryos contributed to the survival of mice derived from embryonic stem (ES) cells by tetraploid embryo complementation. Twenty-five newborns were produced by aggregation of hybrid ES cells and tetraploid embryos with different genetic backgrounds. These newborns were entirely derived from ES cells judged by microsatellite DNA (A specific sequence of DNA bases or nucleotides that contains mono, di, tri or tetra repeats) and coat colour phenotype and germline transmission. Fifteen survived to adulthood while seven died of respiratory failure. All newborns were derived from outbred or hybrid tetraploid aggregates and no newborns were from the inbreds. Our results demonstrate that the genetic heterozygosity, fitness of tetraploid embryos and fitness of ES cells are crucial parameters influencing survival of mice derived from ES cells by tetraploid embryo aggregation. In addition, this method represents a simple and efficient procedure for immediate generation of targeted mouse mutants from genetically modified ES cell clones, in contrast to the standard protocol, which involves the production of chimeras and several breeding steps.

Simple and efficient production of mice derived from embryonic stem cells aggregated with tetraploid embryos.

Six newly derived hybrid mouse embryonic stem (ES) cell lines and two inbred ES cell lines were tested for their ability to produce completely ES cell-derived mice by aggregation of ES cells with tetraploid embryos. Forty-five ES cell-tetraploid pups were generated from six hybrid ES cell lines and no pups from two inbred ES cell lines. These pups were found to have increased embryonic and placental weights than control mice. Twenty-two pups survived to adulthood and produced normal offsprings, and the other 23 pups died of several reasons including respiratory distress, abdomen ulcer-like symptoms, and foster failure. The 22 adult ES cell-tetraploid mice were completely ES cell-derived as judged by coat color and germline transmission, only two of them was found to have tetraploid component in liver, blood, and lung as analyzed by microsatellite loci. Our data suggested that genetic heterozygosity is a crucial factor for postnatal survival of ES cell-tetraploid mice, and tetraploid embryo aggregation using hybrid ES cells is a simple and efficient procedure for immediate generation of targeted mouse mutants from genetically modified ES cell clones, in contrast to the standard protocol, which involves the production of chimeras and several breeding steps.

The proteasomal inhibitor MG132 increases the efficiency of mouse embryo production after cloning by electrofusion.

In this study, we cloned mice from ES cells by a post-electrofusion MG132 treatment and improved development of cloned embryos with a sequential cultivation protocol. When 5 microM MG132, a proteasome inhibitor, were used to treat the reconstructed embryos, the capacity of in vitro development, implantation and full-term development were significantly improved. Blastocyst formation rates of the reconstructed embryos from X4 ES cells (F1 strain derived from C57BL/6 x 129sv) and J1 ES cells obtained with or without MG132 treatment were 66.9% and 26.6%, and 66.1% and 34.5% respectively (P < 0.05). A total of 146 two-cell embryos cloned from X4 ES cells with MG132 treatment were transferred to recipients, and five cloned pups (3.4%) were born, of which four survived. When the same numbers of two-cell embryos cloned from X4 ES cells without MG132 treatment were transferred, however, no live-born mice were obtained. When embryos cloned from J1 ES cells without MG132 treatment were cultured in KSOM medium for 54 h followed by culture in CZB medium containing 5.6 mM glucose for 42 h, the blastocyst rate was significantly higher than when they were cultured in KSOM continuously for 96 h (34.5% vs 17.1%). However, sequential cultivation did not improve the development of embryos cloned with MG132 treatment and that of parthenotes. In conclusion, MG132 treatment increased the developmental potential of reconstructed mouse embryos, and sequential cultivation improved development of the embryos cloned by electrofusion without MG132 treatment.