Quantifying growing versus non-growing ovarian follicles in the mouse.

BACKGROUND: A standard histomorphometric approach has been used for nearly 40 years that identifies atretic (e.g., dying) follicles by counting the number of pyknotic granulosa cells (GC) in the largest follicle cross-section. This method holds that if one pyknotic granulosa nucleus is seen in the largest cross section of a primary follicle, or three pyknotic cells are found in a larger follicle, it should be categorized as atretic. Many studies have used these criteria to estimate the fraction of atretic follicles that result from genetic manipulation or environmental insult. During an analysis of follicle development in a mouse model of Fragile X premutation, we asked whether these 'historical' criteria could correctly identify follicles that were not growing (and could thus confirmed to be dying). METHODS: Reasoning that the fraction of mitotic GC reveals whether the GC population was increasing at the time of sample fixation, we compared the number of pyknotic nuclei to the number of mitotic figures in follicles within a set of age-matched ovaries. RESULTS: We found that, by itself, pyknotic nuclei quantification resulted in high numbers of false positives (improperly categorized as atretic) and false negatives (improperly categorized intact). For preantral follicles, scoring mitotic and pyknotic GC nuclei allowed rapid, accurate identification of non-growing follicles with 98% accuracy. This method most often required the evaluation of one follicle section, and at most two serial follicle sections to correctly categorize follicle status. For antral follicles, we show that a rapid evaluation of follicle shape reveals which are intact and likely to survive to ovulation. CONCLUSIONS: Combined, these improved, non-arbitrary methods will greatly improve our ability to estimate the fractions of growing/intact and non-growing/atretic follicles in mouse ovaries.

Granulosa cell and oocyte mitochondrial abnormalities in a mouse model of fragile X primary ovarian insufficiency.

STUDY HYPOTHESIS: We hypothesized that the mitochondria of granulosa cells (GC) and/or oocytes might be abnormal in a mouse model of fragile X premutation (FXPM). STUDY FINDING: Mice heterozygous and homozygous for the FXPM have increased death (atresia) of large ovarian follicles, fewer corpora lutea with a gene dosage effect manifesting in decreased litter size(s). Furthermore, granulosa cells (GC) and oocytes of FXPM mice have decreased mitochondrial content, structurally abnormal mitochondria, and reduced expression of critical mitochondrial genes. Because this mouse allele produces the mutant Fragile X mental retardation 1 (Fmr1) transcript and reduced levels of wild-type (WT) Fmr1 protein (FMRP), but does not produce a Repeat Associated Non-ATG Translation (RAN)-translation product, our data lend support to the idea that Fmr1 mRNA with large numbers of CGG-repeats is intrinsically deleterious in the ovary. WHAT IS KNOWN ALREADY: Mitochondrial dysfunction has been detected in somatic cells of human and mouse FX PM carriers and mitochondria are essential for oogenesis and ovarian follicle development, FX-associated primary ovarian insufficiency (FXPOI) is seen in women with FXPM alleles. These alleles have 55-200 CGG repeats in the 5' UTR of an X-linked gene known as FMR1. The molecular basis of the pathology seen in this disorder is unclear but is thought to involve either some deleterious consequence of overexpression of RNA with long CGG-repeat tracts or of the generation of a repeat-associated non-AUG translation (RAN translation) product that is toxic. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Analysis of ovarian function in a knock-in FXPM mouse model carrying 130 CGG repeats was performed as follows on WT, PM/+, and PM/PM genotypes. Histomorphometric assessment of follicle and corpora lutea numbers in ovaries from 8-month-old mice was executed, along with litter size analysis. Mitochondrial DNA copy number was quantified in oocytes and GC using quantitative PCR, and cumulus granulosa mitochondrial content was measured by flow cytometric analysis after staining of cells with Mitotracker dye. Transmission electron micrographs were prepared of GC within small growing follicles and mitochondrial architecture was compared. Quantitative RT-PCR analysis of key genes involved in mitochondrial structure and recycling was performed. MAIN RESULTS AND THE ROLE OF CHANCE: A defect was found in follicle survival at the large antral stage in PM/+ and PM/PM mice. Litter size was significantly decreased in PM/PM mice, and corpora lutea were significantly reduced in mice of both mutant genotypes. Mitochondrial DNA copy number was significantly decreased in GC and metaphase II eggs in mutants. Flow cytometric analysis revealed that PM/+ and PM/PM animals lack the cumulus GC that harbor the greatest mitochondrial content as found in wild-type animals. Electron microscopic evaluation of GC of small growing follicles revealed mitochondrial structural abnormalities, including disorganized and vacuolar cristae. Finally, aberrant mitochondrial gene expression was detected. Mitofusin 2 (Mfn2) and Optic atrophy 1 (Opa1), genes involved in mitochondrial fusion and structure, respectively, were significantly decreased in whole ovaries of both mutant genotypes. Mitochondrial fission factor 1 (Mff1) was significantly decreased in PM/+ and PM/PM GC and eggs compared with wild-type controls. LIMITATIONS, REASONS FOR CAUTION: Data from the mouse model used for these studies should be viewed with some caution when considering parallels to the human FXPOI condition. WIDER IMPLICATIONS OF THE FINDINGS: Our data lend support to the idea that Fmr1 mRNA with large numbers of CGG-repeats is intrinsically deleterious in the ovary. FXPM disease states, including FXPOI, may share mitochondrial dysfunction as a common underlying mechanism. LARGE SCALE DATA: Not applicable. STUDY FUNDING AND COMPETING INTERESTS: Studies were supported by NIH R21 071873 (J.J./G.H), The Albert McKern Fund for Perinatal Research (J.J.), NIH Intramural Funds (K.U.), and a TUBITAK Research Fellowship Award (B.U.). No conflict(s) of interest or competing interest(s) are noted.

The roles of parathyroid hormone-like hormone during mouse preimplantation embryonic development.

Parathyroid hormone-like hormone (PTHLH) was first identified as a parathyroid hormone (PTH)-like factor responsible for humoral hypercalcemia in malignancies in the 1980s. Previous studies demonstrated that PTHLH is expressed in multiple tissues and is an important regulator of cellular and organ growth, development, migration, differentiation, and survival. However, there is a lack of data on the expression and function of PTHLH during preimplantation embryonic development. In this study, we investigated the expression characteristics and functions of PTHLH during mouse preimplantation embryonic development. The results show that Pthlh is expressed in mouse oocytes and preimplantation embryos at all developmental stages, with the highest expression at the MII stage of the oocytes and the lowest expression at the blastocyst stage of the preimplantation embryos. The siRNA-mediated depletion of Pthlh at the MII stage oocytes or the 1-cell stage embryos significantly decreased the blastocyst formation rate, while this effect could be corrected by culturing the Pthlh depleted embryos in the medium containing PTHLH protein. Moreover, expression of the pluripotency-related genes Nanog and Pou5f1 was significantly reduced in Pthlh-depleted embryos at the morula stage. Additionally, histone acetylation patterns were altered by Pthlh depletion. These results suggest that PTHLH plays important roles during mouse preimplantation embryonic development.

Roles of MAPK and spindle assembly checkpoint in spontaneous activation and MIII arrest of rat oocytes.

Rat oocytes are well known to undergo spontaneous activation (SA) after leaving the oviduct, but the SA is abortive with oocytes being arrested in metaphase III (MIII) instead of forming pronuclei. This study was designed to investigate the mechanism causing SA and MIII arrest. Whereas few oocytes collected from SD rats at 13 h after hCG injection that showed 100% of mitogen-activated protein kinase (MAPK) activities activated spontaneously, all oocytes recovered 19 h post hCG with MAPK decreased to below 75% underwent SA during in vitro culture. During SA, MAPK first declined to below 45% and then increased again to 80%; the maturation-promoting factor (MPF) activity fluctuated similarly but always began to change ahead of the MAPK activity. In SA oocytes with 75% of MAPK activities, microtubules were disturbed with irregularly pulled chromosomes dispersed over the spindle and the spindle assembly checkpoint (SAC) was activated. When MAPK decreased to 45%, the spindle disintegrated and chromosomes surrounded by microtubules were scattered in the ooplasm. SA oocytes entered MIII and formed several spindle-like structures by 6 h of culture when the MAPK activity re-increased to above 80%. While SA oocytes showed one Ca(2+) rise, Sr(2+)-activated oocytes showed several. Together, the results suggested that SA stimuli triggered SA in rat oocytes by inducing a premature MAPK inactivation, which led to disturbance of spindle microtubules. The microtubule disturbance impaired pulling of chromosomes to the spindle poles, caused spindle disintegration and activated SAC. The increased SAC activity reactivated MPF and thus MAPK, leading to MIII arrest.

Protein profile changes during porcine oocyte aging and effects of caffeine on protein expression patterns.

It has been shown that oocyte aging critically affects reproduction and development. By using proteomic tools, in the present study, changes in protein profiles during porcine oocyte aging and effects of caffeine on oocyte aging were investigated. By comparing control MII oocytes with aging MII oocytes, we identified 23 proteins that were up-regulated and 3 proteins that were down-regulated during the aging process. In caffeine-treated oocytes, 6 proteins were identified as up-regulated and 12 proteins were identified as down-regulated. A total of 38 differentially expressed proteins grouped into 5 regulation patterns were determined to relate to the aging and anti-aging process. By using the Gene Ontology system, we found that numerous functional gene products involved in metabolism, stress response, reactive oxygen species and cell cycle regulation were differentially expressed during the oocyte aging process, and most of these proteins are for the first time reported in our study, including 2 novel proteins. In addition, several proteins were found to be modified during oocyte aging. These data contribute new information that may be useful for future research on cellular aging and for improvement of oocyte quality.

Glucose metabolism in mouse cumulus cells prevents oocyte aging by maintaining both energy supply and the intracellular redox potential.

Inhibiting oocyte postovulatory aging is important both for healthy reproduction and for assisted reproduction techniques. Some studies suggest that glucose promotes oocyte meiotic resumption through glycolysis, but others indicate that it does so by means of the pentose phosphate pathway (PPP). Furthermore, although pyruvate was found to prevent oocyte aging, the mechanism is unclear. The present study addressed these issues by using the postovulatory aging oocyte model. The results showed that whereas the oocyte itself could utilize pyruvate or lactate to prevent aging, it could not use glucose unless in the presence of cumulus cells. Glucose metabolism in cumulus cells prevented oocyte aging by producing pyruvate and NADPH through glycolysis and PPP. Whereas PPP was still functioning after inhibition of glycolysis, the glycolysis was completely inactivated after inhibition of PPP. Addition of fructose-6-phosphate, an intermediate product from PPP, alleviated oocyte aging significantly when the PPP was totally inhibited. Lactate prevented oocyte aging through its lactate dehydrogenase-catalyzed oxidation to pyruvate, but pyruvate inhibited oocyte aging by its intramitochondrial metabolism. However, both lactate and pyruvate required mitochondrial electron transport to prevent oocyte aging. The inhibition of oocyte aging by both PPP and pyruvate involved regulation of the intracellular redox status. Together, the results suggest that glucose metabolism in cumulus cells prevented oocyte postovulatory aging by maintaining both energy supply and the intracellular redox potential and that) glycolysis in cumulus cells might be defective, with pyruvate production depending upon the PPP for intermediate products.

Survivin is a critical regulator of spindle organization and chromosome segregation during rat oocyte meiotic maturation.

Survivin is a novel member of the inhibitor of apoptosis gene family that bear baculoviral IAP repeats (BIRs), whose physiological roles in regulating meiotic cell cycle need to be determined. Confocal microscopy was employed to observe the localization of survivin in rat oocytes. At the germinal vesicle (GV) stage, survivin was mainly concentrated in the GV. At the prometaphase I (pro-MI) and metaphase I (MI) stage, survivin was mainly localized at the kinetochores, with a light staining detected on the chromosomes. After transition to anaphase I or telophase I stage, survivin migrated to the midbody, and signals on the kinetochores and chromosomes disappeared. At metaphase II (MII) stage, survivin became mainly localized at the kinetochores again. Microinjection of oocytes with anti-survivin antibodies at the beginning of the meiosis, thus blocking the normal function of survivin, resulted in abnormal spindle assembly, chromosome segregation and first polar body emission. These results suggest that survivin is involved in regulating the meiotic cell cycle in rat oocytes.

Short-term preservation of porcine oocytes in ambient temperature: novel approaches.

The objective of this study was to evaluate the feasibility of preserving porcine oocytes without freezing. To optimize preservation conditions, porcine cumulus-oocyte complexes (COCs) were preserved in TCM-199, porcine follicular fluid (pFF) and FCS at different temperatures (4°C, 20°C, 25°C, 27.5°C, 30°C and 38.5°C) for 1 day, 2 days or 3 days. After preservation, oocyte morphology, germinal vesicle (GV) rate, actin cytoskeleton organization, cortical granule distribution, mitochondrial translocation and intracellular glutathione level were evaluated. Oocyte maturation was indicated by first polar body emission and spindle morphology after in vitro culture. Strikingly, when COCs were stored at 27.5°C for 3 days in pFF or FCS, more than 60% oocytes were still arrested at the GV stage and more than 50% oocytes matured into MII stages after culture. Almost 80% oocytes showed normal actin organization and cortical granule relocation to the cortex, and approximately 50% oocytes showed diffused mitochondria distribution patterns and normal spindle configurations. While stored in TCM-199, all these criteria decreased significantly. Glutathione (GSH) level in the pFF or FCS group was higher than in the TCM-199 group, but lower than in the non-preserved control group. The preserved oocytes could be fertilized and developed to blastocysts (about 10%) with normal cell number, which is clear evidence for their retaining the developmental potentiality after 3d preservation. Thus, we have developed a simple method for preserving immature pig oocytes at an ambient temperature for several days without evident damage of cytoplasm and keeping oocyte developmental competence.

Fertilization in vitro with spermatozoa from different mice increased variation in the developmental potential of embryos compared to artificial parthenogenetic activation.

Although successful embryo development is dependent upon genetic and epigenetic contributions from both the male and female, the male potential to adversely affect embryo development has been scarcely studied. It is unclear whether the sperm variation among different males would affect the outcome of oocyte evaluation by embryo development following fertilization. In the present study, variation in the developmental potential of mouse embryos was first compared between in vitro fertilization with epididymal spermatozoa from different males and Sr(2+) parthenogenetic activation using oocytes of different qualities, and then the effect of male on fertilization and embryo development was examined using randomly chosen oocytes and spermatozoa from cauda epididymidis, vas deferens or electro-ejaculates. Rates of fertilization and blastocyst formation were significantly higher with spermatozoa from cauda epididymidis or vas deferens than with ejaculated spermatozoa. Rates of embryonic development differed significantly between different males, but not between different ejaculates of the same male. Analysis of standard errors of means and coefficients of variance indicated that as long as multiple males were involved, the variation in oocyte fertilization/activation and blastocyst formation was always higher after fertilization than after Sr(2+) parthenogenetic activation whether spermatozoa were collected from epididymidis, vas deferens or ejaculates and regardless of oocyte qualities. It is concluded that (1) epididymal mouse spermatozoa fertilize more oocytes than ejaculated spermatozoa under identical experimental conditions; (2) like farm animals, the mice also show a remarkable male effect on the developmental potential of in vitro produced embryos although they are supposed to be less genetically diverse; (3) parthenogenetic activation is recommended for assessment of oocyte quality to exclude the effect of male.

Dynamic changes of germinal vesicle chromatin configuration and transcriptional activity during maturation of rabbit follicles.

Dynamic changes in configuration of germinal vesicle (GV) chromatin and their effects on oocyte transcriptional activity and developmental competence were studied in rabbit oocytes. The results indicated that global condensation of GV chromatin, rather than the formation of a perinucleolar ring, would better reflect the global transcriptional repression and developmental competence of oocytes. Gonadotropins, by promoting large-scale chromatin remodeling, regulate oocyte transcriptional activity and developmental competence.

Effects of heat stress during in vitro maturation on cytoplasmic versus nuclear components of mouse oocytes.

The objectives of this study were to investigate the effect of heat stress during in vitro maturation on the developmental potential of mouse oocytes and to determine whether the deleterious effect was on the nuclear or cytoplasmic component. While rates of oocyte nuclear maturation (development to the metaphase II stage) did not differ from 37 to 40 degrees C, rates for blastocyst formation decreased significantly as maturation temperature increased from 38.5 to 39 degrees C. Chromosome spindle exchange showed that while blastocyst formation did not differ when spindles matured in vivo or in vitro at 37, 40 or 40.7 degrees C were transplanted into in vivo matured cytoplasts, no blastocyst formation was observed when in vivo spindles were transferred into the 40 degrees C cytoplasts. While oocytes reconstructed between 37 degrees C ooplasts and 37 or 40 degrees C karyoplasts developed into 4-cell embryos at a similar rate, no oocytes reconstituted between 40 degrees C ooplasts and 37 degrees C spindles developed to the 4-cell stage. Immunofluorescence microscopy revealed impaired migration of cortical granules and mitochondria in oocytes matured at 40 degrees C compared with oocytes matured at 37 degrees C. A decreased glutathione/GSSG ratio was also observed in oocytes matured at 40 degrees C. While spindle assembling was normal and no MAD2 was activated in oocytes matured at 37 or 40 degrees C, spindle assembling was affected and MAD2 was activated in some of the oocytes matured at 40.7 degrees C. It is concluded that 1) oocyte cytoplasmic maturation is more susceptible to heat stress than nuclear maturation, and 2) cytoplasmic rather than nuclear components determine the pre-implantation developmental capacity of an oocyte.

Developmental and hormonal regulation of cumulus expansion and secretion of cumulus expansion-enabling factor (CEEF) in goat follicles.

The objective of this article was to study the developmental and hormonal regulation of cumulus expansion and secretion of cumulus expansion-enabling factor (CEEF) in goat follicles. M-199 medium was conditioned for 24 hr with cumulus-denuded oocytes (DOs), oocytectomized complexes (OOXs), or mural granulosa cells (MGCs) from goat follicles of different sizes. Mouse OOXs and eCG were added to culture drops of the conditioned medium and cumulus expansion was scored at 18 hr of culture to assess CEEF production. While mouse OOXs did not expand, goat OOXs underwent full cumulus expansion when cultured in nonconditioned eCG-supplemented M-199 medium. When cultured in nonconditioned medium containing 10% follicular fluid (FF) from goat medium (2-4 mm) and small (0.8-1.5 mm) follicles, 71-83% mouse OOXs expanded; but expansion rates decreased (P < 0.05) at either lower or higher FF concentrations. FF from large (5-6 mm) follicles did not support mouse OOX expansion at any concentrations. While medium conditioned with DOs from follicles of all the three sizes supported expansion of 80-90% mouse OOXs, medium conditioned with mature DOs had no effect. While cumulus cells from follicles of all the three sizes secreted CEEF in the absence of gonadotropins, MGCs from large follicles became gonadotropin dependent for CEEF production. Both FSH and LH stimulated CEEF production by large follicle MGCs, but FSH had a shorter half-life than LH to expand mouse OOXs. Few meiosis-incompetent goat oocytes from small follicles underwent cumulus expansion when cultured in medium conditioned with goat DOs or cocultured with goat COCs from medium follicles. It is concluded that (1) goat cumulus expansion is independent of the oocyte; (2) the limited CEEF activity in FF from large follicles was due mainly to the inability of MGCs in these follicles to secret the factor in absence or short supply of gonadotropins; (3) the cumulus expansion inability of the meiosis incompetent goat oocytes was due to the inability of their cumulus cells to respond to rather than to produce CEEF.

Cumulus cells accelerate aging of mouse oocytes by secreting a soluble factor(s).

Control of oocyte aging in vitro is important for both human-assisted reproduction and animal embryo technologies because fertilization or artificial activation of aged oocytes results in abnormal development. Interactions between somatic and germ cells are also an important issue in current biological research. The role of cumulus cells (CCs) in maturation, ovulation, and fertilization of oocytes has been extensively studied, yet little is known about their role in oocyte aging. Although our previous study has shown that CCs accelerate the aging progression of mouse oocytes, the mechanism by which CCs accelerate oocyte aging is unknown. In this study, cumulus-denuded mouse oocytes (DOs) were co-cultured with cumulus-oocyte complexes (COCs) or CC monolayer or cultured in medium conditioned with these cells and changes in the susceptibility to activating stimuli and in MPF activity of oocytes were evaluated after different aging treatments. The results showed that culture with or in medium conditioned with COCs or CC monolayer promoted activation of DOs, indicating that a soluble factor is responsible for the aging-promoting effect. The in vivo and in vitro-matured DOs did not differ in responsiveness to the aging-promoting factor (APF). Heat shock did not accelerate oocyte aging unless in the presence of CCs. The production of APF was not affected by the age or maturation system of COCs, but increased with their density and duration of culture. The results strongly suggest that CCs accelerated oocyte aging by secreting a soluble APF into the medium. Further analysis showed that the APF was heat labile but stable to freezing, it had a threshold effective concentration and can be depleted by DOs.

Selection of oocytes for in vitro maturation by brilliant cresyl blue staining: a study using the mouse model.

Selecting oocytes that are most likely to develop is crucial for in vitro fertilization and animal cloning. Brilliant cresyl blue (BCB) staining has been used for oocyte selection in large animals, but its wider utility needs further evaluation. Mouse oocytes were divided into those stained (BCB+) and those unstained (BCB-) according to their ooplasm BCB coloration. Chromatin configurations, cumulus cell apoptosis, cytoplasmic maturity and developmental competence were compared between the BCB+ and BCB- oocytes. The effects of oocyte diameter, sexual maturity and gonadotropin stimulation on the competence of BCB+ oocytes were also analyzed. In the large- and medium-size groups, BCB+ oocytes were larger and showed more surrounded nucleoli (SN) chromatin configurations and higher frequencies of early atresia, and they also gained better cytoplasmic maturity (determined as the intracellular GSH level and pattern of mitochondrial distribution) and higher developmental potential after in vitro maturation (IVM) than the BCB- oocytes. Adult mice produced more BCB+ oocytes with higher competence than the prepubertal mice when not primed with PMSG. PMSG priming increased both proportion and developmental potency of BCB+ oocytes. The BCB+ oocytes in the large-size group showed more SN chromatin configurations, better cytoplasmic maturity and higher developmental potential than their counterparts in the medium-size group. It is concluded that BCB staining can be used as an efficient method for oocyte selection, but that the competence of the BCB+ oocytes may vary with oocyte diameter, animal sexual maturity and gonadotropin stimulation. Taken together, the series of criteria described here would allow for better choices in selecting oocytes for better development.

Effects of delayed excision of oviducts/ovaries on mouse oocytes and embryos.

To achieve the best and reproducible results of experiments, effects of delayed excision of oviducts/ovaries on mouse ovarian/ovulated oocytes and embryos have been studied. Oviducts/ovaries were excised at different times after death of mice and effects of the postmortem interval on ovarian/ovulated oocytes and embryos were analyzed. When oviduct excision was delayed 10 min, many ovulated oocytes lysed or underwent in vitro spontaneous activation, and this postmortem effect aggravated with the extension of postmortem interval and oocyte aging. Oocytes from different mouse strains responded differently to delayed oviduct removal. Delayed oviduct excision did not cause lysis of zygotes or embryos but compromised their developmental potential. When ovaries were excised at 30 min after death, percentages of atretic follicles increased while blastocyst cell number declined significantly after oocyte maturation in vitro. Preservation of oviducts in vitro, in intact or opened abdomen at different temperatures and histological analysis of oviducts from different treatments suggested that toxic substance(s) were secreted from the dying oviducts which induced oocyte lysis and spontaneous activation and both this effect itself and the sensitivity of oocytes to this effect was temperature dependent. It is concluded that a short delay of oviduct/ovary removal had marked detrimental effects on oocytes and embryos. This must be taken into account in experiments using oocytes or embryos from slaughtered animals. The data may also be important for estimation of the time of death in forensic medicine and for rescue of oocytes from deceased valuable or endangered mammals.

Effects of cell cycle status on the efficiency of liposome-mediated gene transfection in mouse fetal fibroblasts.

Methods for cell cycle synchronization of mouse fetal fibroblast cells (MFFCs) were first selected and optimized. When MFFCs were cooled at 5 C for different periods of time, the highest percentage of cells at the G0/G1 phase (75.4+/-2.9%), with 3.5+/-0.3% of apoptotic cells, was achieved after 5 h of treatment. Extended cooling increased the number of apoptotic cells significantly. When MFFCs were treated with different concentrations of roscovitine (ROS) for different periods of time, the highest percentage of G0/G1 cells (83.5+/-1.8%), with 9.2+/-0.6% apoptotic cells, was obtained after exposure to 10 microM ROS for 24 h. When the cells were cooled at 5 C for 5 h followed by incubation in 10 microM ROS for 12 h, 83.6+/-1.9% were synchronized at the G0/G1 stage, with 3.6% undergoing apoptosis. Cell cycle progression was then observed after release of the MFFCs from different synchronization blocks. The highest percentages of S and G2/M cells (81% and 75%) were achieved at 12 and 20 h, respectively, after release of the MFFCs from the cooling plus ROS treatment, and these percentages were significantly higher than those obtained after release from the cooling or ROS alone blocks. Finally, MFFCs were transfected with pEGFP-N1 plasmid at the peak of the G0/G1, S, and G2/M phases, respectively, after release from the different blocks and both the transient and stable transfection efficiencies were determined. The GFP gene expression was greatly enhanced when transfection was performed at the time when most cells were at the G2/M stage after release from cooling, ROS alone, and cooling plus ROS treatments. Statistical analysis revealed a close correlation between the rate of G2/M cells and the transient and stable GFP gene expression efficiencies. Together, the results indicated that (a) the best protocol for cell cycle synchronization of MFFCs was a 5-h cooling at 5 C followed by incubation in 10 microM ROS for 12 h which produced both a high rate of synchronization in the G0/G1 phase with acceptable apoptosis and a high rate of G2/M cells after release; and (b) that the cell cycle status had marked effects on the efficiency of liposome-mediated transfection in MFFCs, with the highest transfection efficiency obtained in cells at the G2/M stage.

[Factors affecting liposome-mediated gene transfection of mouse somatic cells].

We studied the effects of the amount of liposome and plasmid, exposure time of cells to the liposome-plasmid complexes, number of cell passages and cell types on GFP gene transfection of mouse somatic cells. The maximal GFP transgene expression (30.7%) was achieved when mouse fetal fibroblast cells (MFFC) at 70%-90% confluence of passage 3 were exposed for 6 h to the complexes of 4 microg liposome (LipofectAMINE) and 0.3 microg plasmid (pEGFP-N1). Under these conditions, we compared the effect of the number (from primary to 15) of passages on the transfection efficiency of MFFC. The transfection efficiency of MFFC was 10.0%, 28.9% and 7.2% at the primary, 3rd and 15th passage, respectively, which indicated that the transfection efficiency decreased with passaging. When MFFC, mouse oviductal epithelial cells (MOEC) and mouse granulosa cells (MGC) were transfected at passage 3, the transfection efficiency was 27.8%, 13.7% and 14.2%, respectively, under the described transfection conditions. When the cell cycle stages of different cell types at transfection were examined, it was found that 17.2% of MFFC, 8.7% of MOEC and 9.9% of MGC were at M phases of the cell cycle. Examination of the cell cycle stages of MFFC at different passages showed that MFFC at the third passage had the highest percentage of M cells and the percentage decreased afterwards. This suggested that the transfection efficiency was correlated with the percentages of cells at M phase, and provided essential data for improvement of the transfection efficiency.

[Cryopreservation of mouse embryos in ethylene glycol-based solutions: a search for the optimal and simple protocols].

Although ethylene glycol (EG) has been widely used for embryo cryopreservation in domestic animals, few attempts were made to use this molecule to freeze mouse and human embryos. In the few studies that used EG for slow-freezing of mouse and human embryos, complicated protocols for human embryos were used, and the protocols need to be simplified. Besides, freezing mouse morula with EG as a cryoprotectant has not been reported. In this paper, we studied the effects of embryo stages, EG concentration, duration and procedure of equilibration, sucrose supplementation and EG removal after thawing on the development of thawed mouse embryos, using the simple freezing and thawing procedures for bovine embryos. The blastulation and hatching rates (81.92% +/- 2.24% and 68.56% +/- 2.43%, respectively) of the thawed late compact morulae were significantly (P < 0.05) higher than those of embryos frozen-thawed at other stages. When mouse late compact morulae were frozen with different concentrations of EG, the highest rates of blastocyst formation and hatching were obtained with 1.8mol/L EG. The blastulation rate was significantly higher when late morulae were equilibrated in 1.8 mol/L EG for 10 min prior to freezing than when they were equilibrated for 30 min, and the hatching rate of embryos exposed to EG for 10 min was significantly higher than that of embryos exposed for 20 and 30 min. Both rates of blastocyst formation and hatching obtained with two-step equilibration were higher (P < 0.05) than with one-step equilibration in 1.8 mol/L EG. Addition of sucrose to the EG-based solution had no beneficial effects. On the contrary, an increased sucrose level (0.4 mol/L) in the solution impaired the development of the frozen-thawed embryos. In contrast, addition of 0.1 mol/L sucrose to the propylene glycol (PG)-based solution significantly improved the development of the frozen-thawed embryos. Elimination of the cryoprotectant after thawing did not improve the development of the thawed embryos. The cell numbers were less (P < 0.05) in blastocysts developed from the thawed morulae than in the in vivo derived ones. In summary, embryo stage, EG concentration, duration and procedure of equilibration and sucrose supplementation had marked effects on development of the thawed mouse embryos, and a protocol for cryopreservation of mouse embryos is recommended in which the late morulae are frozen in 1.8 mol/L EG using the simple freezing and thawing procedures of bovine embryos after a two-step equilibration and the embryos can be cultured or transferred without EG removal after thawing.

Cortical granules behave differently in mouse oocytes matured under different conditions.

BACKGROUND: To better understand the differences between in vivo (IVO) and in vitro (IVM) matured oocytes, we studied the chronological changes in cortical granule (CG) distribution and nuclear progression during maturation, and the competence of CG release and embryo development of mouse oocytes matured under different conditions. METHODS: Oocytes matured in vivo or in different culture media were used and CG distribution and release were assessed by fluorescein isothiocyanate-labelled Lens culinaris agglutinin and laser confocal microscopy. RESULTS: Tempos of nuclear maturation and CG redistribution were slower, and competence for CG exocytosis, cleavage and blastulation were lower in the IVM oocytes than in the IVO oocytes. These parameters also differed among oocytes matured in different culture media. Hypoxanthine (HX, 4 mM) blocked germinal vesicle breakdown (GVBD), postponed CG migration and prevented CG-free domain (CGFD) formation. Cycloheximide (CHX) facilitated both GVBD and CG migration, but inhibited CGFD formation. The presence of serum in maturation media enhanced CG release after aging or activation of oocytes. Maintenance of germinal vesicle intact for some time by a trace amount (0.18 mM) of HX was beneficial to oocyte cytoplasmic maturation. CONCLUSION: CGs behaved differently in mouse oocytes matured under different conditions, and cytoplasmic maturity was not fully achieved in the IVM oocytes.

Parthenogenetic activation of mouse oocytes by strontium chloride: a search for the best conditions.

Strontium has been successfully used to induce activation of mouse oocytes in nuclear transfer and other experiments, but the optimum treatment conditions have not been studied systematically. When cumulus-free oocytes were treated with 10mM SrCl(2) for 0.5-5h, activation rates (88.4+/-4.1 to 91.2+/-2.7%) did not differ (mean+/-S.E.; P>0.2), but rate of blastulation (57.3+/-3.5%) and cell number per blastocyst (45.0+/-2.4) were the highest after treatment for 2.5h. When treated with 1-20mM SrCl(2) for 2.5h, the activation rate and cell number per blastocyst were higher (P<0.02) after 10mM SrCl(2) treatment than other treatments. The best activation and development were obtained with Ca(2+)-free Sr(2+) medium, but the activation rate was low (37.7+/-1.6%) in Ca(2+)-containing medium. Activation rates were the same, regardless of the presence or absence of cytochalasin B (CB) in the activating medium, but the blastulation rate was higher (P<0.001) in the presence of CB. Only 70% of the cumulus-enclosed oocytes were activated and 10% blastulated after a 10 min exposure to 1.6mM SrCl(2), and many lysed, with increased intensity of Sr(2+) treatment. The presence of CB in SrCl(2) medium markedly reduced lysis of cumulus-enclosed oocytes. Media M16 and CZB did not differ when used as activating media. Only 10.5% of the oocytes collected 13 h post hCG were activated by Sr(2+) treatment alone, with 34% blastulating, but rates of activation and blastulation increased (P<0.001) to 94 and 60%, respectively, when they were further treated with 6-dimethylaminopurine (6-DMAP). The total and ICM cell numbers were less (P<0.001) in parthenotes than in the in vivo fertilized embryos. In conclusion, the concentration and duration of SrCl(2) treatment and the presence or absence of CB in activating medium and cumulus cells had marked effects on mouse oocyte activation and development. To obtain the best activation and development, cumulus-free oocytes collected 18 h post hCG should be treated for 2.5h with 10mM SrCl(2) in Ca(2+)-free medium supplemented with 5 microg/mL of CB.