Xin-Yong Liu

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.

Maternal-restraint stress increases oocyte aneuploidy by impairing metaphase I spindle assembly and reducing spindle assembly checkpoint proteins in mice.

ABSTRACT Studies in both humans and animals suggest detrimental effects of psychological stress on reproduction. Although our recent study shows that maternal-restraint stress diminishes oocyte developmental potential, the mechanism behind this effect is unknown. This prompted us to study the potential role of maternal-restraint stress in the genesis of aneuploidy during meiosis I. At 24 h after equine chorionic gonadotropin injection, mice were subjected to restraint stress for 24 h. After the restraint, some mice were killed to recover immature oocytes for in vitro maturation, while others were injected with human chorionic gonadotropin to recover in vivo matured oocytes. Analysis on chromosome complements of both mature oocytes and parthenotes confirmed that maternal restraint increased aneuploidy in both in vivo and in vitro matured oocytes and that the percentage of aneuploid oocytes were three times higher in the earlier matured oocytes than in the later matured ones. Further observations indicated that maternal restraint 1) impaired metaphase I (MI) spindle assembly while inhibiting MAPK activities, 2) accelerated progression of anaphase I while down-regulating the expression of spindle assembly checkpoint (SAC) proteins, and 3) induced intraoocyte oxidative stress. The following possible model was proposed to explain the results. Maternal-restraint stress increased oocyte aneuploidy by impairing MI spindle assembly and decreasing the SAC. Whereas abnormal spindles would affect centromere attachments, a reduction in SAC would accelerate the anaphase I progression. Failure of centromere attachment, together with the hastened anaphase, would result in nondisjunction of the unattached chromosomes. Furthermore, maternal-restraint stress might also impair spindle assembly and SAC function by inducing intraoocyte oxidative stress, which would then reduce MAPK activity, a critical regulator of microtubule assembly and the establishment and maintenance of the SAC during oocyte maturation.

Effects of species and cellular activity of oviductal epithelial cells on their dialogue with co-cultured mouse embryos

An efficient co-culture system, especially with oviductal or uterine epithelial cells, is important not only for the production of high quality embryos, but also for the study of the molecular dialogue between embryos and their maternal environment. Although mouse embryos have been co-cultured successfully with oviductal epithelial cells (OECs) from several species, studies on the effects of species and functionality of OECs are few. Reports concerning the necessity of direct contact between the embryo and OECs and about the culture of mouse embryos in medium conditioned with heterologous OECs have been controversial. In this study, pronuclear embryos from Kunming mice, characterized by an obvious two-cell block in vitro, were co-cultured with mouse, goat, and chick OECs. The functionality of OECs was determined by analyzing the cell cycle, apoptosis, the numbers of mitochondria and cilia, and the ability both to support embryonic development and to remove hypoxanthine from the culture medium. The necessity of direct contact between OECs and embryos was studied by repeated renewal of culture medium with fresh conditioned medium, the culture of embryos in plastic wells connected by tunnels to wells with OEC monolayers, and the co-culture of embryos separated from OECs by a filter. Both goat and chick OECs supported mouse embryonic development, but their embryotrophic lifespan was shorter than that of the mouse OECs. Whereas media conditioned with mouse OECs supported mouse embryonic development satisfactorily, medium conditioned with goat OECs supported little development. Immediate dialogue between heterologous OECs and embryos was essential for efficient co-culture, whereas direct contact between the two cell types was not; neither dialogue nor contact was needed between isologous OECs and embryos. Embryotrophic activity and the ability to remove hypoxanthine from conditioned medium declined with time after confluence and number of passages of OECs, mainly because of apoptosis and dedifferentiation. Thus, the species and functionality of OECs have profound effects on their molecular dialogue with co-cultured embryos, and efficient co-culture depends upon both positive and negative conditioning.

Effects of activation on functional aster formation, microtubule assembly, and blastocyst development of goat oocytes injected with round spermatids.

A systematic study was conducted on round spermatids (ROS) injection (ROSI) using the goat model. After ROSI, the oocytes were treated or not with ionomycin (ROSI+I and ROSI-I, respectively) and compared with intracytoplasmic sperm injection (ICSI). After ROSI-I, most oocytes were arrested with premature chromatin condensation and few oocytes formed pronuclei. In contrast, most oocytes formed pronuclei after ROSI+I. Some ROS were observed to form asters that organized a dense microtubule network after ROSI+I, but after ROSI-I, no ROS asters were observed. Whereas most of the oocytes showed Ca(2+) rises and a significant decline in maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activities after ROSI+I, no such changes were observed after ROSI-I. Due to the lack of Ca(2+) oscillations after ROSI-I, oocytes were injected with more ROS. Interestingly, different from the results observed in a single ROS injection, injection with four ROS effectively activated oocytes by inducing typical Ca(2+) oscillations. Whereas ROSI+I oocytes and ICSI oocytes both showed extensive microtubule networks, no such a network was observed in parthenogenetic oocytes. Together, the results suggest that goat ROS is not able to trigger intracellular Ca(2+) rises and thus to inhibit MPF and MAPK activities, but artificial activation improved fertilization and development of ROSI goat oocytes. Goat ROS can organize functional microtubular asters in activated oocytes. A ROS-derived factor(s) may be essential for organization of a functional microtubule network to unite pronuclei. Goat centrosome is of paternal origin because both ROS and sperm asters organized an extensive microtubule network after intra-oocyte injection.

Cyclin B1 turnover and the mechanism causing insensitivity of fully grown mouse oocytes to cycloheximide inhibition of meiotic resumption

Cyclin B1 turnover and the insensitivity of fully-grown mouse oocytes to cycloheximide (CHX) inhibition of germinal vesicle breakdown (GVBD) were examined by assaying GVBD and cyclin B1 levels after treatment of oocytes with various combinations of eCG and CHX. Whereas over 95% of oocytes underwent GVBD after culture for 24 h with CHX alone, only 10% did so after culture with CHX + eCG (P < 0.05). In addition, preculture with eCG alone had no effect, but preculture with eCG + CHX prevented GVBD during a second culture with CHX alone. Therefore, we inferred that eCG delayed GVBD long enough for CHX inhibition of protein synthesis to allow cyclin B1 to decrease below a threshold where GVBD became dependent upon its de novo synthesis. However, western blot revealed no cyclin B1 synthesis, but cyclin B1 degradation, as long as GVs were maintained intact with eCG. Regarding the function of CHX in preculture without protein synthesis to block subsequent GVBD, whereas eCG delayed GVBD for only 3 h, CHX had an ongoing effect that further postponed GVBD, thus allowing cyclin B1 to decrease below the threshold. When oocytes precultured with eCG + CHX were further cultured without eCG and CHX, cyclin B1 first decreased but then, because of the ongoing effects of CHX, increased to a level sufficient to induce GVBD. The content of P34Cdc2 was not altered under any of the culture conditions (P > 0.05). We concluded that insensitivity of mouse germinal vesicle (GV) oocytes to CHX was due to the presence of sufficient cyclin B1, and that cyclin B1 level in such oocytes was maintained by an equilibrium between synthesis and degradation.

Role of calcium-sensing receptor in regulating spontaneous activation of postovulatory aging rat oocytes

Abstract Mechanisms for postovulatory aging (POA) of oocytes and for spontaneous activation (SA) of rat oocytes are largely unknown. Expression of calcium-sensing receptor (CaSR) in rat oocytes and its role in POA remain unexplored. In this study, expression of CaSR in rat oocytes aging for different times was detected by immunofluorescencemicroscopy, and western blotting and the role of CaSR in POA was determined by observing the effects of regulating its activity on SA susceptibility and cytoplasmic calcium levels. The results showed that CaSR was expressed in rat oocytes. Oocytes recovered 19 h post human chorionic gonadotropin (hCG) injection were more susceptible to SA and expressed more functional CaSR than oocytes recovered 13 h after hCG injection, although both expressed the same level of total CaSR protein. Treatment with CaSR antagonist significantly suppressed cytoplasmic calcium elevation and SA of oocytes. Activation of Na-Ca2+ exchangerwith NaCl inhibited SA to a greater extent than suppression of CaSR with NPS-2143, suggesting that calcium sources other than CaSR-controlled channels contributed to the elevation of cytoplasmic calcium. Treatment with T- or L-type calcium channel blockers significantly reduced SA. Suppression of all calcium channels tested reduced SA to minimum. It is concluded that the level of CaSR functional dimer protein, but not that of the total CaSR protein, was positively correlated with the SA susceptibility during POA of rat oocytes confirming that CaSR is involved in POA regulation. Blocking multiple calcium channels might be a better choice for efficient control of SA in rat oocytes. Summary Sentence Calcium-sensing receptor is involved in regulating postovulatory aging of oocytes, and blocking multiple calcium channels may be used for efficient control of spontaneous activation in rat oocytes.