Yan-Guang Wu

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.

Interactive effects of granulosa cell apoptosis, follicle size, cumulus–oocyte complex morphology, and cumulus expansion on the developmental competence of goat oocytes: a study using the well-in-drop culture system

Using a well-in-drop (WID) oocyte/embryo culture system that allows identification of follicular origin, we have investigated the effects of granulosa cells (GCs) apoptosis, follicle size, cumulus-oocyte complexes (COCs) morphology, and cumulus expansion on the developmental competence of goat oocytes matured and cultured individually following parthenogenetic activation. The WID system supported oocyte maturation and embryo development to a level similar to the conventional group system. The majority of goat oocytes acquired competence for development up to the 8-16 cell stage in follicles larger than 2 mm, but did not gain the ability to form morula/blastocyst (M/Bs) until follicles larger than 3 mm in diameter. The extent of atresia affected M/Bs formation. This effect varied according to the follicle size. Cumulus expansion increased with follicle size and decreased with increasing incidence of GCs apoptosis. Oocyte developmental potential was also correlated with cumulus expansion. Regardless of the degree of follicle atresia, 73-84% of the floating cells in the follicular fluid (FF) underwent apoptosis. Correlation between floating cell density in FF and oocyte developmental potency suggests the possibility to use the floating cell density as a simple and non-invasive marker for oocyte quality. It is concluded that the developmental potential of an oocyte is determined by multifactor interactions, and multiple factors must be considered together to accurately predict the quality of an oocyte.

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.

Pyruvate prevents aging of mouse oocytes

Inhibiting oocyte aging is important not only for healthy reproduction but also for the success of assisted reproduction techniques. Although our previous studies showed that cumulus cells accelerated aging of mouse oocytes, the underlying mechanism is unknown. The objective of this paper was to study the effects of pyruvate and cumulus cells on mouse oocyte aging. Freshly ovulated mouse cumulus-oocyte complexes (COCs) or cumulus-denuded oocytes (DOs) were cultured in Chatot-Ziomek-Bavister (CZB) medium or COC-conditioned CZB medium supplemented with different concentrations of pyruvate before being examined for aging signs and developmental potential. Pyruvate supplementation to CZB medium decreased rates of ethanol-induced activation in both COCs and DOs by maintaining their maturation-promoting factor activities, but more pyruvate was needed for COCs than for DOs. Addition of pyruvate to the COC-conditioned CZB also alleviated aging of DOs. Observations on cortical granules, level of BCL2 proteins, histone acetylation, intracellular concentration of glutathione, and embryo development all confirmed that pyruvate supplementation inhibited aging of mouse oocytes. It is concluded that the aging of mouse oocytes, facilitated by culture in COCs, can be partially prevented by the addition of pyruvate to the culture medium.

Demecolcine-Assisted Enucleation of Goat Oocytes: Protocol Optimization, Mechanism Investigation, and Application to Improve the Developmental Potential of Cloned Embryos

Although demecolcine-assisted enucleation has been performed successfully in porcine and cattle, the mechanism and protocol optimization of chemically assisted enucleation need further investigation. The present study optimized the protocol for goat oocyte enucleation and demonstrated that a 30-min treatment with 0.8 ng/mL demecolcine-induced cytoplasmic protrusions in over 90% of the oocytes. Rates of enucleation, cell fusion, and blastocyst formation were significantly higher after demecolcine-assisted than after blind aspiration enucleation, although differences in rates of live births remain to be unequivocally determined between the two treatments. The ability to form protrusions decreased significantly as spindles became less organized in aged oocytes and the oocytes with a poor cumulus expansion. More than 93% of the demecolcine-induced protrusions persisted for 2 h in the absence of cytochalasin B (CB) but most disappeared within 30 min of CB treatment. The spindle disintegrated, an actin-rich ring formed around the chromosome mass and the MAP kinase activity increased significantly after demecolcine treatment. When oocytes with induced protrusions were treated with CB, however, the contractile ring disappeared, the spindle reintegrated, and both MPF and MAP kinase activities decreased significantly. It is concluded that (1) cytoplasmic protrusions can be induced in goat oocytes with a very low concentration of demecolcine; (2) oocyte selection and enucleation can be achieved simultaneously with demecolcine treatment; and (3) an interactive effect between the MAP kinase, MPF, microfilaments and microtubules might be implicated in the control of cytoplasmic protrusion formation after demecolcine treatment.

Mouse Cumulus-Denuded Oocytes Restore Developmental Capacity Completely When Matured with Optimal Supplementation of Cysteamine, Cystine, and Cumulus Cells

Our objectives were to study how cysteamine, cystine, and cumulus cells (CCs), as well as oocytes interact to increase oocyte intracellular glutathione (GSH) and thereby to establish an efficient in vitro maturation system for cumulus-denuded oocytes (DOs). Using M16 that contained no thiol as maturation medium, we showed that when supplemented alone, neither cystine nor cysteamine promoted GSH synthesis of mouse DOs, but they did when used together. Although goat CCs required either cysteamine or cystine to promote GSH synthesis, mouse CCs required both. In the presence of cystine, goat CCs produced cysteine but mouse CCs did not. Cysteamine reduced cystine to cysteine in cell-free M16. When TCM-199 that contained 83 μM cystine was used as maturation medium, supplementation with cysteamine alone had no effect, but supplementation with 100 μM cysteamine and 200 μM cystine increased blastulation of DOs matured with CC coculture to a level as high as achieved in cumulus-surrounded oocytes (COCs). Similar numbers of young were produced after two-cell embryos from mouse COCs or CC-cocultured DOs matured with optimal thiol supplementation were transferred to pseudopregnant recipients. It is concluded that 1) mouse CCs can use neither cysteamine nor cystine to promote GSH synthesis, but goat CCs can use either one; 2) goat CCs promote mouse oocyte GSH synthesis by reducing cystine to cysteine, but how they use cysteamine requires further investigation; and 3) mouse DOs can use neither cystine nor cysteamine for GSH synthesis, but they restore developmental capacity completely when matured in the presence of optimum supplementation of cysteamine, cystine, and CCs.

Caffeine can be used for oocyte enucleation.

The removal of chromosomes from recipient oocytes is one of the key steps in nuclear transfer cloning. Although microtubule interrupters have been successfully used for oocyte enucleation, their potential side effect on oocyte developmental potential should be considered, and less harmful drugs should be explored for chemical-assisted enucleation. Based on our previous findings that any maturation promoting factor-activating agent induces ooplasmic protrusion without disrupting microtubules, we have studied the feasibility to use caffeine or MG132 for chemical-assisted enucleation. Experiments using goat oocytes showed that treatments for 30 min with 1-mM caffeine or 5-μM MG132-induced ooplasmic protrusions in about 85% of the oocytes, a percentage similar to that achieved with optimal demecolcine treatment. Rates of enucleation, cell fusion and in vitro blastulation were similar among caffeine, MG132, and demecolcine enucleation but significantly higher than blind aspiration. Furthermore, neither rates of pregnancy on days 90 and 120 nor the general rate of live births/embryos transferred differed significantly (p > 0.05) between caffeine and demecolcine enucleation. Although oocytes treated with caffeine did not retract protrusions until 2 h, many oocytes treated with MG132 withdrew protrusions as early as 0.5 h after treatment. The optimal treatment to induce ooplasmic protrusion in 75% pig oocytes was 8-mM caffeine for 60 min. Mouse oocytes responded poorly to demecolcine or caffeine with less than 40% forming inconspicuous protrusions following optimal treatments. It is concluded that caffeine can be used for enucleation of goat and pig oocytes with similar results as demecolcine, and live kids were born after caffeine-assisted enucleation.

MPF Governs the Assembly and Contraction of Actomyosin Rings by Activating RhoA and MAPK during Chemical-Induced Cytokinesis of Goat Oocytes

The interplay between maturation-promoting factor (MPF), mitogen-activated protein kinase (MAPK) and Rho GTPase during actin-myosin interactions has yet to be determined. The mechanism by which microtubule disrupters induce the formation of ooplasmic protrusion during chemical-assisted enucleation of mammalian oocytes is unknown. Moreover, a suitable model is urgently needed for the study of cytokinesis. We have established a model of chemical-induced cytokinesis and have studied the signaling events leading to cytokinesis using this model. The results suggested that microtubule inhibitors activated MPF, which induced actomyosin assembly (formation of ooplasmic protrusion) by activating RhoA and thus MAPK. While MAPK controlled actin recruitment on its own, MPF promoted myosin enrichment by activating RhoA and MAPK. A further chemical treatment of oocytes with protrusions induced constriction of the actomyosin ring by inactivating MPF while activating RhoA. In conclusion, the present data suggested that the assembly and contraction of the actomyosin ring were two separable steps: while an increase in MPF activity promoted the assembly through RhoA-mediated activation of MAPK, a decrease in MPF activity triggered contraction of the ring by activating RhoA.