De-Qiang Miao

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.

Cumulus Cells Accelerate Aging of Mouse Oocytes

Abstract The role of cumulus cells (CCs) that surround oocytes in maturation, ovulation, and fertilization has been extensively studied, yet little is known about their role in oocyte aging. Although early studies have shown that when ovulated oocytes are aged in vitro displayed similar morphological alterations as those aged in vivo, a recent study found that vitro culture of mouse oocytes retarded oocyte aging. The objective of this study was to test the hypothesis that CCs would accelerate oocyte aging. During in vitro aging with CCs of both in vivo-matured and in vitro-matured mouse oocytes, activation rates increased, whereas the maturation-promoting factor (MPF) activity decreased significantly as during in vivo aging of the ovulated oocytes. During aging after denudation of CCs, however, activation rates of both in vivo-matured and in vitro-matured oocytes remained low and the MPF activity decreased much more slowly compared to that of oocytes aged with CCs. Although many oocytes aged in vivo and in vitro with CCs showed a partial cortical granule (CG) release, very few cumulus-free oocytes released their CGs during in vitro aging. When denuded oocytes were cultured with cumulus-oocyte-complexes at a 1:2 ratio or on a CC monolayer, activation rates increased, while MPF activity decreased significantly. The results strongly suggested that CCs accelerated the aging progression of both in vivo-matured and in vitro-matured mouse oocytes.

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.

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 Ca2+ rise, Sr2+-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.

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.

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.

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. Mol. Reprod. Dev. 75: 1387–1395, 2008.

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 Sr2+ 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 Sr2+ 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. Mol. Reprod. Dev. 76: 239–245, 2009.