Influence of meiotic inhibition by butyrolactone-I during germinal vesicle stage on the ability of porcine oocytes to be activated by electric stimulation after nuclear maturation

Butyrolactone-I (BL-I) is a specific inhibitor of cyclin-dependent kinases and prevents germinal vesicle breakdown (GVBD) in porcine oocytes. This study first focused on the effect of BL-I on the time course of GVBD and progression to metaphase II (MII) in oocytes after the removal of BL-I. When porcine oocytes were treated with 20 microM BL-I for 28 h, the intervals taken to undergo GVBD and progress to MII were 4-8 h and 16-20 h after washing out BL-I, respectively. These intervals were both approximately 8 h behind those of the control oocytes. When the BL-I treatment was performed for 20 h, instead of 28 h, there were no differences in the timing or frequency of progression to MII between the BL-I treatment and control groups. To determine whether the cytoplasmic maturation was the same, the ability of oocytes to form a female pronucleus in response to an electric stimulus was examined. When oocytes were stimulated at 28 h after the removal of BL-I, the rate of pronucleus formation was significantly lower in oocytes treated with BL-I than in untreated oocytes, despite the fact that both groups reached MII at a similar time. When the electric stimulus was given at 36 h after the removal of BL-I, oocytes had a pronucleus formation rate comparable to that of control oocytes. Therefore, the findings suggest that BL-I treatment of porcine oocytes at the germinal vesicle stage affects not only the progression of meiosis, but also the events involved in cytoplasmic maturation.     

Pronuclear synchronization and nuclear morphology of mature and in vitro matured oocytes in the rat: An ultrastructural study

Summary The objective of this study was to evaluate synchronous and asynchronous pronucleus (PN) formation and the related patterns of juxtapositional nucleolus (n) formation in immature (prophase I [PI] and metaphase I [MI]) and mature (metaphase II [MII]) oocytes after fertilization, both ultrastructurally and at the level of light microscope. A single dose of 15 IU gonadotrophin was injected subcutaneously to twenty four 26-wk-old, female Wistar rats to induce ovulation. Human chorionic gonadotrophin (4 IU) was administered 40 h later, and after 4–6 h the ovaries were dissected, and the oocytes were aspirated. A total of 214 rat oocytes were classified according to a maturation index as follows: group I, 80 PI oocytes; group II, 50 MI oocytes; and group III, 84 MII oocytes. Immature oocytes were in vitro matured for 18–36 h. Spermatozoa were acquired by microepididymal sperm aspiration and processed using swim-up technique. Intracytoplasmic sperm injection was performed on mature oocytes after 2 h of incubation and on in vitro matured (IVM) oocytes 4 h after maturation. Pronuclear synchronization [both pronucleases (PNs) centrally located, equal sized, with equal numbers and sizes of juxtapositional nucleoli (Nn)] was observed in fertilized oocytes. Asynchronous PN formation (diversity between male and female PNs, related to dimensions, localization, and the number of Nn) in groups I, II, and III was found in 75, 86, and 47% of preembryos, respectively. There was a significant difference of synchronous pronuclear formation between mature and IVM oocytes (P<0.05). In IVM oocytes, asynchronous PN formation is high, and juxtapositional pronucleolar patterns are observed to be low by transmission electron microscope (TEM).     

Evolution of the acquisition of fertilization competence and polyspermy blocks during meiotic maturation

In many animals, fully grown oocytes are arrested at prophase of meiosis I. Before or after ovulation/spawning, a secondary arrest occurs at metaphase of meiosis I or II (MI or II, respectively). MI arrest in the ovary is released after spawning, and is followed by fertilization, whereas MI and MII arrest after ovulation are released by fertilization. Insemination of isolated oocytes from the ovaries at an inappropriate time increases the rate of polyspermy, indicating that ovaries provide the proper environment for acquisition of the polyspermy blocks and the development of competence to be fertilized normally. Due to MI arrest in the ovaries or MI/MII arrest after ovulation/spawning, the fertilizable period can be elongated. Thus, MI and MII arrest may play a role in maintaining the cell-cycle phases to enable normal fertilization. Here, the evolution of fertilization timing is discussed.     

Pronuclear formation in starfish eggs inseminated at different stages of meiotic maturation: correlation of sperm nuclear transformations and activity of the maternal chromatin

Changes in sperm nuclei incorporated into starfish, Asterina miniata, eggs inseminated at different stages of meiosis have been correlated with the progression of meiotic maturation. A single, uniform rate of sperm expansion characterized eggs inseminated at the completion of meiosis. In oocytes inseminated at metaphase I and II the sperm nucleus underwent an initial expansion at a rate comparable to that seen in eggs inseminated at the pronuclear stage. However, in oocytes inseminated at metaphase I, the sperm nucleus ceased expanding by meiosis II and condensed into chromosomes which persisted until the completion of meiotic maturation. Concomitant with the formation and expansion of the female pronucleus, sperm chromatin of oocytes inseminated at metaphase I enlarged and developed into male pronuclei. Condensation of the initially expanded sperm nucleus in oocytes inseminated at metaphase II was not observed. Instead, the enlarged sperm nucleus underwent a dramatic increase in expansion commensurate with that taking place with the maternal chromatin to form a female pronucleus. Fusion of the relatively large female pronucleus and a much smaller male pronucleus was observed in eggs fertilized at the completion of meiotic maturation. In oocytes inseminated at metaphase I and II, the male and female pronuclei, which were similar in size, migrated into juxtaposition, and as separate structures underwent prophase. The chromosomes in each pronucleus condensed, intermixed, and became aligned on the metaphase palate of the mitotic spindle in preparation for the first cleavage division. These observations demonstrate that the time of insemination with respect to the stage of meiotic maturation has a significant effect on sperm nuclear transformations and pronuclear morphogenesis.     

In vitro maturation of domestic dog oocytes cultured in advanced preantral and early antral follicles

Initial studies in our laboratory demonstrated that a large proportion of domestic dog advanced preantral (APAN) and early antral (EAN) follicles contained grown oocytes that had acquired the dense cytoplasmic lipid characteristic of preovulatory oocytes. The objective of this study was to assess nuclear maturation of those oocytes after in vitro culture. Both APAN and EAN follicles (152 to 886 microns in diameter) were isolated from ovaries by treatment with collagenase and DNase. The follicles were cultured in Dulbecco's Modified Eagle's medium/nutrient mixture F-12 Ham culture medium supplemented with 20% (v/v) fetal bovine serum (FBS), 2 mM L-glutamine, 1% (v/v) antibiotic-antimycotic, 1 microgram FSH/ml, 10 IU hCG/ml and 1 microgram estradiol/ml. Within each group (APAN or EAN), control follicles were not cultured (0 h), and 2 to 12 follicles per well were incubated under a humidified atmosphere of 5% CO2 in air at 37 degrees C for 24, 48 or 72 h. After 24 h of culture, significantly more (5.3%, 20/374; P < 0.05) oocytes from APAN follicles reached the metaphase I to metaphase II stages (MI to MII) than the percentage of control follicles observed at 0 h (0.9%, 3/318). Continued culture resulted in a further increase (P < 0.05) in the percentage of oocytes reaching MI to MII by 48 h (11.5%, 47/407), which remained unchanged at 72 h (9.9%, 40/404). The percentage of oocytes from EAN follicles reaching MI to MII did not significantly increase after 24 h of culture. However, there was an increase (P < 0.05) by 48 h of culture (8.7%, 11/126), which remained unchanged at 72 h (7.5%, 8/106). These results show that dog oocytes cultured within advanced preantral and early antral follicles in vitro are competent to resume meiosis to the metaphase stage.     

Cytogenetic evaluation of in vitro matured bovine oocytes collected from ovaries of individual donors

Oocytes were collected after slaughter by aspiration from pairs of ovaries of individual donors. A total of 656 oocytes was selected for IVM from 74 pairs of ovaries (8.9 oocytes per pair, ranging between 1 and 25). The oocytes were matured in droplets of maturation medium (TCM-199 medium supplemented with 20% estrous cow serum (ECS), 50 microg/ml gentamycin, 10 microg/ml FSH, 1 microg/ml estradiol-17beta). Cytogenetic analysis of 348 oocytes showed 79 at the first metaphase (MI; 22.7%, 79 348 ), 11 at the first telophase (TI; 3.2%, 11/348 ), and 258 at the second metaphase (MII; 74.1% 258/348 ). Significant differences (P < 0.01) were shown among the donors regarding the number of oocytes selected for IVM and the number of oocytes matured for IVF.     

In vitro culture of mouse GV oocytes and preantral follicles isolated from ovarian tissues cryopreserved by vitrification

Cryopreservation of ovarian tissues containing many immature oocytes occurs in both gamete/embryo research and clinical medicine. Using vitrification, we studied factors related to meiosis after cryopreservation using the COCs (cumulus oocyte complexes) and preantral follicles obtained from cryopreserved ovarian tissues. COCs were isolated and cultured for 17 approximately 19 hr. Thereafter, Metaphase II stage (MII stage) oocytes and fertilized oocytes after IVF were observed at a rate of 76.5% and 60.0%, respectively. Preantral follicles (100 approximately 130 microm in diameter) were isolated and cultured in alpha MEM containing hFSH, ITS, and FBS. HCG and EGF were added to the media to stimulate ovulation on the 12th day of culture. The survival rates of the follicles obtained from the frozen/thawed ovaries were 66.4%. After 12 days of culture, the diameter of the follicles isolated from fresh (620.2 +/- 11.3 microm) and frozen/thawed ovaries (518.7 +/- 15.1 microm) differed as did the estradiol concentrations (3474.2 +/- 159 pg/ml vs. 1508.2 +/- 134 pg/ml). After in vitro ovulation, MII stage oocytes were observed in 84.5% of the fresh group and 60.5% of the frozen/thawed group while the fertilization rate was 74.2% and 53.5%, respectively. These studies demonstrate that cryopreservation of mouse ovarian tissues by vitrification did not affect the oocyte's ability to undergo meiosis. Thus, this technique may become a powerful tool for the preservation of the female gamete.     

The effect of in-situ nerve growth factor from different biological sources on the reinitiation of mouse oocyte meiotic maturation in culture and on parthenogenetic activation

We studied the capacity of mouse oocytes to complete meiotic maturation in vitro and form the female pronucleus upon parthenogenetic activation by cycloheximide, in response to a single injection into the mouse ovaries in situ of a purified fraction of 2.5 S NGF from mouse submaxillary glands and beta-NGF from bovine sperm. Injection of NGF from both sources at 10 ng/ml with subsequent incubation of the ovaries for 1 h increased the capacity of matured oocytes for parthenogenetic formation of the pronucleus. The frequency of pronucleus formation in both "naked oocyte" and oocytes surrounded by the cumulus cells was four times that in the control.     

Effect of telophase enucleation on bovine somatic nuclear transfer

Telophase enucleation has been proven to be an efficient method for preparing recipient cytoplasts in bovine embryonic nuclear transfer (2, 11). This research was designed to study in vitro development of bovine oocytes containing transferred somatic cell nuclei, reconstructed by using enucleated in vitro-matured oocytes 32 h of age at telophase II stage as recipient cytoplasts, compared with those 24 h of age at metaphase II stage. Two protocols for donor cell injection were adopted, i.e., subzonal injection (SUZI) and intracytoplasmic injection (ICI). Bovine oviduct epithelial cells (BOECs) and bovine cumulus cells (BCCs) from an adult cow were used as nuclear donors for these experiments. In SUZI groups, the fusion rate of donor cells, both BOECs and BCCs, with MII enucleated oocytes were higher than those with TII enucleated oocytes (54% vs. 41% and 53% vs. 39%, respectively; P<0.05), but the development rates to morula plus blastocyst stage in MII groups were lower than those in TII groups (22% vs. 39% and 21% vs. 41%, respectively; P<0.05). In ICI groups, about 26% of enucleated MII oocytes injected with BOECs or BCCs cleaved and only small parts of them developed to blastocyst stage (4% and 3%, respectively; P>0.05). When BOECs or BCCs were intracytoplasmically injected into oocytes enucleated at TII stage, no blastocyst was formed in either donor cell group and no cleavage occurred in BOEC group. Our data demonstrated that telophase enucleation is beneficial to early embryo development when bovine somatic nuclei are transferred by subzonal injection. However, it is harmful when donor cells are directly injected into the cytoplast of the enucleated oocytes.     

Influence of reproductive status on in vitro oocyte maturation in dogs

In the bitch, oocytes need 48-72 h to complete post-ovulatory maturation to the metaphase II stage in the isthmus of the oviduct, an interval similar to that found in in vitro studies. The effect of estrous cycle stage on in vitro meiotic competence of dog oocytes has been described in several studies. However, there are no reports evaluating the possible effects of pyometra or pregnancy on subsequent potential of oocytes recovered from such females to undergo in vitro maturation.In this study, immature cumulus-oocyte complexes (COCs) were recovered from fresh excised domestic dog ovaries in various reproductive states. The donor females were classified into groups based on stage of the estrous cycle: follicular (proestrus or estrus), luteal (diestrus) or anestrus or at the clinical conditions of pregnancy and pyometra. Grades 1 and 2 oocytes were cultured in vitro at 37 degrees C in TCM-199, supplemented with 25 mM Hepes/l (v/v), and with 10% heat inactived estrous cow serum (ECS), 50 microg/ml gentamicin, 2.2 mg/ml sodium carbonate, 22 microg/ml pyruvic acid, 1.0 microg/ml estradiol, 0.5 microg/ml FSH and 0.03 IU/ml hCG. The nuclear maturation rate was evaluated at 72 h of incubation under Hoechst 33342 (10 microg/ml) staining for fluorescence microscopy. There was no statistical difference in nuclear progression to the MII stage among the various reproductive states (follicular phase, 5.4%; diestrus, 4.2%; anestrus, 4.4%; pyometra, 8.1% and pregnancy, 4.7%). Resumption of meiosis was 24.6% at the follicular phase, 19.6% for diestrus, 16.4% for anestrus, 37.1% for pyometra and 29.2% for pregnancy. Positive and higher numbers of residue above the expected value were observed for the pyometra and pregnancy conditions at the metaphase/anaphase I (MI/AI) stages.Our results indicate that in vitro nuclear maturation of dogs oocytes is not influenced by the in vivo reproductive status of the female. The quality of the oocyte is a more reliable indicator of its potential for meiotic maturation in vitro than the hormonal environment of the donor female at the time of oocyte retrieval.     

Rotation of meiotic spindle is controlled by microfilaments in mouse oocytes

The completion of meiosis requires the spatial and temporal coordination of cytokinesis and karyokinesis. During meiotic maturation, many events, such as formation, location, and rotation of the meiotic spindle as well as chromosomal movement, polar body extrusion, and pronuclear migration, are dependent on regulation of the cytoskeleton system. To study functions of microfilaments in meiosis, we induced metaphase II (MII) mouse oocytes to resume meiosis by in vitro fertilization or parthenogenetic activation, and we treated such oocytes with cytochalasin B (CB). The changes of the meiotic spindle, as visualized in preparations stained for beta-tubulin and chromatin, were observed by fluorescent confocal microscopy. The meiotic spindle of MII oocytes was observed to be parallel to the plasmalemma. After meiosis had resumed, the spindle rotated to the vertical position so that the second polar body could be extruded into the perivitelline space. When meiosis resumed and oocytes were treated with 10 micro g/ml of CB, the spindle rotation was inhibited. Consequently, the oocyte formed an extra pronucleus instead of extruding a second polar body. These results indicate that spindle rotation is essential for polar body extrusion; it is the microfilaments that play a crucial role in regulating rotation of the meiotic spindle.     

Spontaneous and sperm-induced activation of oocytes in LT/Sv strain mice

The oocytes of LT/Sv strain mice are unique in that a high proportion of them (∼40% in this study) are ovulated before reaching metaphase of the second meiotic division (metaphase II). The remaining oocytes of LT/Sv mice are ovulated at metaphase II, as in other strains of mice. When recently ovulated oocytes were cultured in vitro for 11–12 h, those ovulated at metaphase II remained at this stage, whereas those ovulated at metaphase of the first meiotic division (metaphase I) commonly resumed meiosis during in vitro aging. These oocytes extrude the polar body and form a diploid pronucleus. This oocyte activation is not coupled with cortical granule exocytosis. The oocytes ovulated at metaphase II are fully capable of normal fertilization, whereas those ovulated at metaphase I are not. Approximately 50% of metaphase I oocytes penetrated by spermatozoa remain at this stage, and sperm nuclei frequently undergo premature chromosome condensation. Only 13% of spermpenetrated metaphase I oocytes formed a diploid female pronucleus and a haploid male pronucleus by 4 h after insemination. These results demonstrate that the two types of ovulated LT/Sv oocytes have different potentials to undergo either spontaneous or sperm-induced activation.     

Cleavage development of bovine oocytes fertilized by sperm injection

Whole in vitro capacitated bovine spermatozoa were microinjected directly into the ooplasm of in vitro matured bovine oocytes in order to determine whether oocytes fertilized by sperm injection could undergo normal pronuclear formation and cleavage development. Immature oocytes recovered from follicles (2-5 mm) of unstimulated ovaries were cultured for 24-25 h in modified TCM 199 medium supplemented with heat-treated day 20 cow serum, luteinizing hormone (LH), and estradiol 17-B. In vitro capacitated, frozen-thawed spermatozoa were injected into the ooplasm, and the injected oocytes were cultured for an additional 24-28 h. Twenty-one percent (21/101) of the sperm-injected oocytes contained a sperm within the ooplasm; however, only 2% (2/101) cleaved. The remaining oocytes either did not contain a sperm or had degenerated. After oocyte activation induced by a 5 min incubation in 1 microM A23187, sperm nuclear decondensation occurred in the A23187-activated, injected oocytes but not in the unactivated, injected controls (37% vs. 0% after 3 h). Those injected, activated oocytes that contained a male pronucleus also exhibited a female pronucleus and second polar body. Furthermore, a significantly higher number (28%, 6/21) of the injected, activated oocytes cleaved to a two- to four-cell stage after 48 h than did the injected, unactivated oocytes (4%). These results indicate that, unlike hamster and rabbit oocytes, bovine oocytes are not sufficiently stimulated by the injection procedure to complete meiosis, but, upon activation by calcium ionophore, they will undergo normal-appearing cleavage development following fertilization by sperm injection.     

Mouse Oocyte Maturation: Meiotic Checkpoints

Mouse oocytes at different stages of maturation were fused together and the ensuing cell cycle events were analyzed with the objective of identifying checkpoints in meiosis. Fusion of maturing oocytes just undergoing germinal vesicle breakdown (GVBD) induces PCC (premature chromosome condensation) but no spindle formation in immature (GV) partner oocytes. On the other hand, fusion of metaphase I (MI) oocytes containing spindles to GV oocytes induces both PCC and spindle formation in the immature partner. Thus, while molecules required for condensation are present throughout metaphase, those involved in spindle formation are absent in early M-phase. Oocytes cultured for 6 h—early metaphase I (i.e., 2 h before the onset of anaphase I)—and then fused to anaphase-telophase I (A-TI) fusion partners block meiotic progression in the more advanced oocytes and induce chromatin dispersal on the spindle. By contrast, oocytes cultured for 8 h (late MI) before fusion to A-TI partners are driven into anaphase by signals from the more advanced oocytes and thereafter advance in synchrony to telophase I. When early (10 h) or late (12 h) metaphase II oocytes were fused to A-TI partners the signals generated from early MII oocytes block the anaphase to telophase I transition and induce a dispersal of A-TI chromosomes along the spindle. On the other hand, late MII oocytes respond to A-TI signals by exiting from the MII block and undergoing the A-TII transition. Moreover, the oocytes in late MI are not arrested in this stage and progress without any delay through A-TI to MII when fused to metaphase II partners. The signals from the less-developed partner force the MII oocyte through A-TII to MIII. In total, these studies demonstrate that the metaphase period is divided into at least three distinct phases and that a checkpoint in late metaphase controls the progress of meiosis in mammalian oocytes.     

Kinetics of nuclear maturation and protein profiles of oocytes from prepubertal and adult cattle during in vitro maturation

The aim of this present study was to compare the kinetics of nuclear maturation between calf and cow oocytes in order to determine if there are differences between the 2 groups which could explain their disparate developmental capacity. The constitutive and neosynthetic protein patterns of cow and calf oocytes and of their corresponding cumulus cells were also compared during in vitro maturation. A total of 397 calf oocytes and 406 cow oocytes was matured in M199 + 10 ng/mL EGF. The first group of oocytes (n = 30) was immediately fixed and stained after removal from the follicle, and represent 0 h. The remaining oocytes were removed from the maturation medium at 4, 8, 12, 16, 20 and 24 h respectively. Half were denuded, fixed and stained for nuclear status; while the remainder were radiolabeled with methionine-(35S). Immediately after isolation, all the oocytes were at the GV stage. By 8 h, GVBD had occurred in most oocytes (calf: 97%; cow: 100%) and some had reached pro-metaphase I (calf: 49%; cow: 51%). By 12 h, most of the oocytes were at metaphase I (calf: 84%; cow: 94%). By 16 h, 54% of calf oocytes had reached telophase I or beyond compared with 71% of cow oocytes. This difference between the 2 groups became significant by 20 h, with 89% of cow oocytes (P < 0.05) at metaphase II and 71% of calf oocytes. By 24 h of culture, GVBD had occurred in all cases. Most oocytes completed meiosis I and were arrested at metaphase II with the first polar body extruded (calf: 72%; cow: 86%). No differences were noted in the constitutive and the neosynthetic protein profiles of cumulus cells in relation to the age of animal. Changes in neosynthetic protein patterns were observed both in cow and calf cumulus during IVM, and several proteins showed stage-specific synthesis. For the constitutive protein patterns of cow and calf oocytes, there were quantitative (38 and 40 kD) and qualitative (4, 10, 16, 17, 24, 25 and 26 kD) differences between the 2 groups. Only a few differences were observed in neosynthetic proteins between cow and calf oocytes, but there were changes in relation to nuclear status both in cow and calf oocytes. In conclusion, the difference in developmental capacity between cow and calf oocytes may be explained by a difference in the kinetics of nuclear maturation, which was significant at 20 h of culture (with 89% of cow oocytes at metaphase II and 71% of calf oocytes). At the biochemical level, our results indicate that nuclear progression during in vitro maturation of bovine oocytes is linked to changes in protein synthesis by the oocyte itself, while cumulus protein synthesis may either stimulate or modulate the process of oocyte maturation.     

Bovine oocytes treated prior to in vitro maturation with a combination of butyrolactone I and roscovitine at low doses maintain a normal developmental capacity.

Butyrolactone I (BL-I) and Roscovitine (ROS), two specific and potent inhibitors of M-phase promoting factor (MPF) kinase activity, were used to block germinal vesicle breakdown (GVBD) of cattle oocytes. A concentration 6.25 microM BL-I and 12.5 microM ROS blocked over 93.3 +/- 2.5% of oocytes in germinal vesicle (GV) stage during a 24-hr culture period. Following a second 24-hr culture step in maturation medium (IVM) almost all (91.5 +/- 3.0%) inhibited oocytes resumed meiosis and reached the metaphase II (MII) stage. The MII kinetics was different for inhibited and control oocytes. Fifty percent MII was reached at 13-14 hr in BL-I + ROS treated oocytes, compared to 18 hr in control oocytes. Therefore, control oocytes were fertilised (IVF) after 22 hr IVM and inhibited oocytes after 16 or 22 hr IVM. After IVF, percentage of grade 1 freezable embryos on day 7 (D + 7) as well as percentage of blastocyst formation on D + 8 in the group of BL-I + ROS treated oocytes fertilised after 16 hr IVM were higher (P < 0.05) compared with the other experimental group fertilised after 22 hr IVM but not different in comparison with the control. Survival to freezing and thawing of grade 1 embryos frozen on D + 7 was employed as viability criteria and was similar in all groups. Thus, the presence of BL-I + ROS in the prematuration medium of bovine oocytes determines a reversible meiotic block, without compromising their subsequent developmental competence.     

Protein kinase C activity regulates the onset of anaphase I in mouse oocytes

The metaphase-to-anaphase I transition is a key step in the completion of meiosis I. In mouse oocytes, competence to exit metaphase I (MI) is developmentally regulated and typically not acquired until the preovulatory stage. The possible role of protein kinase C (PKC) in regulating this critical transition was assessed in both normal oocytes isolated from small antral follicles (18-day-old B6SJLF1 mice), which have not yet developed the capacity to progress to metaphase II (MII), and also oocytes defective in their ability to exit MI despite development to the preovulatory stage (24-day-old CX8 recombinant inbred strains). In both systems, transient suppression of endogenous PKC activity by treatment with a PKC-specific inhibitor, bisindolylmaleimide I (BIM), promoted the onset of anaphase I in a dose-dependent manner, while activation of PKC with the phorbol ester TPA blocked progression to MII. Following a 2-h incubation with BIM, the majority of oocytes progressed to, and arrested at, MII. The resulting MII oocytes were fertilizable in vitro, showing similar cleavage and blastocyst development rates between BIM treated and untreated controls. Transferred embryos resulted in the development of pups to term in both groups. These data demonstrate that PKC plays an important role in regulating the onset of anaphase I in mouse oocytes. Moreover, it is concluded that oocytes isolated from small antral follicles become blocked at MI due to a PKC-mediated signal, suggesting that acquisition of competence to complete meiosis I involves, in part, the control of PKC activity. Similarly, failure to regulate PKC activity at the preovulatory stage likely promotes arrest at MI.     

In vitro maturation,fertilization and development of domestic cat oocytes recovered from ovaries collected at three stages of the reproductive cycle

This study was conducted to examine the effect of the donor cat's reproductive cycle stage on in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro development of oocytes recovered from ovaries that were collected and stored at 35 degrees C for a short period (1-6 h). Based on the presence or absence of follicles and corpora lutea, the ovarian pairs collected were classified into inactive, follicular, or luteal stages. Nuclear status of 161 cumulus-oocyte complexes (COCs) were examined immediately after recovery; 91.3% of the oocytes were found to be at the immature germinal vesicle (GV) stage, and 3.7% of the oocytes were at metaphase II (MII) stage. The percentage of the oocytes at the GV stage was significantly lower in the follicular stage than in the inactive stage (P < 0.01). Of the oocytes from the follicular stage, 9.1% were at MII stage. After culture for 24 h, however, the proportions of oocytes that reached metaphase I and MII were not different among the reproductive cycle stages of the ovaries collected (P > 0.05). After co-incubation with sperm, 63.1% of oocytes were fertilized, but there were no significant differences among the reproductive cycle stages of the ovaries with respect to the proportions of normal and polyspermic fertilization. However, the number of oocytes reaching cleavage stage and development to the morula and blastocyst stages from follicular stage ovaries were significantly lower (P < 0.05) than those obtained from inactive and luteal stage ovaries. These results indicate that the reproductive cycle stage of donor cat ovaries, stored at 35 degrees C, has no apparent effects on the frequencies of maturation and fertilization of oocytes, but influences developmental competence of the oocytes following IVM or IVF.     

Merotelic kinetochore attachment in oocyte meiosis II causes sister chromatids segregation errors in aged mice

Mammalian oocyte chromosomes undergo 2 meiotic divisions to generate haploid gametes. The frequency of chromosome segregation errors during meiosis I increase with age. However, little attention has been paid to the question of how aging affects sister chromatid segregation during oocyte meiosis II. More importantly, how aneuploid metaphase II (MII) oocytes from aged mice evade the spindle assembly checkpoint (SAC) mechanism to complete later meiosis II to form aneuploid embryos remains unknown. Here, we report that MII oocytes from naturally aged mice exhibited substantial errors in chromosome arrangement and configuration compared with young MII oocytes. Interestingly, these errors in aged oocytes had no impact on anaphase II onset and completion as well as 2-cell formation after parthenogenetic activation. Further study found that merotelic kinetochore attachment occurred more frequently and could stabilize the kinetochore-microtubule interaction to ensure SAC inactivation and anaphase II onset in aged MII oocytes. This orientation could persist largely during anaphase II in aged oocytes, leading to severe chromosome lagging and trailing as well as delay of anaphase II completion. Therefore, merotelic kinetochore attachment in oocyte meiosis II exacerbates age-related genetic instability and is a key source of age-dependent embryo aneuploidy and dysplasia.     

Transplantation of male pronucleus derived from in vitro fertilization of enucleated oocyte into parthenogenetically activated oocyte results in live offspring in mouse

In this study, inter-strain reconstructed embryos were produced by combining the female pronucleus of Kunming mouse (white) with male pronucleus of C57BL/6 strain (black). Metaphase II (MII) oocytes of Kunming mouse were enucleated and the zona pellucida was removed. Then, the enucleated oocytes were inseminated by capacitated sperm of C57BL/6 mouse in vitro. At the same time, MII oocytes of Kunming mouse were artificially activated using strontium chloride solution, which did not contain cytochalasin B. Finally, we removed the male pronucleus derived from C57BL/6 sperm and injected it into a parthenogenetically activated one-pronucleus oocyte by micromanipulation. The reconstructed 2-cell embryos were transplanted into the oviducts of 22 foster mother mice, each receiving about 20 embryos. In the end, seven healthy and live pups were born from one recipient.