Time Of Polar Body Extrusion Research Articles

P-201 Identifying patients benefiting from delayed-matured oocytes insemination

Abstract Study question Are there any predictive factors supporting the decision of inseminating delayed-matured oocytes? Summary answer Patients with ≤59% mature oocytes at retrieval and/or Anti-Mullerian-Hormone (AMH) >2.52 ng/ml have increased chances of obtaining a euploid embryo from delayed-matured oocytes. What is known already Approximately 15% of oocytes retrieved after ovarian stimulation are immature, at metaphase I (MI) or germinal-vesicle (GV) stages at the time of oocyte denudation. Performing IVM in those oocytes could permit an increase on the number of usable embryos. Nevertheless, the utility of delayed-matured oocytes varies greatly among IVF laboratories with relatively low success rates, hence its practice in daily routine might be counter-productive. Determining which patient population could benefit from such strategy is valuable thereof to the clinical practice. Moreover, data comparing euploid rates of embryos derived from delayed-matured oocytes with its mature sibling oocytes are needed. Study design, size, duration This observational study was performed at ART Fertility Clinics, Abu Dhabi, UAE, between January 2019 and June 2021. A total of 5454 cumulus oocytes complexes (COC) were retrieved from 469 ovarian stimulation cycles. Out of the retrieved COCs, 3473 oocytes were immediate at metaphase II (MII-D0), and 915 were delayed-metaphase II oocytes (MII-D1). Participants/materials, setting, methods Patients with primary and secondary infertility undergoing Controlled ovarian stimulation (COS) in standardized protocols for IVF/ICSI treatment were included. Ovum pick up performed 34-36h post final oocyte maturation trigger shot (TS). Insemination was done 39-41h post TS for the MII-D0, while MII-D1 ICSI was performed 63-68h post TS. All cycles were planned for Preimplantation Genetic Testing for Aneuploidies (PGT-A) at blastocyst stage using Next Generation Sequencing (NGS). Main results and the role of chance Fertilization rates significantly differed between MII-D0 and MII-D1 oocytes (69.54% vs 55.96%, p < 0.001, respectively). Blastocyst utilization rates were significantly higher in MII-D0 group compared to MII-D1 group (59.47% vs 18.52%, p < 0.001). However, no difference was observed in the rate of euploid blastocysts between MII-D0 and MII-D1 (46.3% vs 39.0%, p = 0.163). As identified by univariant logistic regression analysis, the following parameters augmented the chances of obtaining at least 1 blastocyst for biopsy when MII-D1 were injected: AMH (OR 1.15, p < 0.001), number of COCs collected (OR: 1.03, P = 0.005), maturation rate on day0 (OR: 0.19, P = 0.001). When the multivariant analysis model was applied, AMH and maturation rate on day0 remained significant factors predicting the success of inseminating delayed-matured oocytes (OR:1.15, [CI:1.00-1.32], p = 0.045); OR:0.06, [CI:0.03-0.31], p < 0.001, respectively), with cut off values of AMH >2.52 ng/ml and maturity rate of ≤ 59%, being identified by ROC analysis. Limitations, reasons for caution ICSI of MII-D1 was performed with the fresh or frozen sperm samples from the previous day. Exact timing of polar body extrusion of delayed-matured MI/GV was not identified. Wider implications of the findings The results of this study might provide guidance to the IVF laboratories for targeting the patient population who would benefit from MII-D1 ICSI without adhering to unnecessary costs and workload. Trial registration number not applicable

Time of morulation and trophectoderm quality are predictors of a live birth after euploid blastocyst transfer: a multicenter study

To investigate whether the morphodynamic characterization of a euploid blastocyst's development allows a higher prediction of a live birth after single-embryo-transfer (SET). Observational cohort study conducted in two phases: training and validation. Private invitro fertilization centers. Euploid blastocysts: 511 and 319 first vitrified-warmed SETs from 868 and 546 patients undergoing preimplantation genetic testing for aneuploidies (PGT-A) in the training and validation phase, respectively. Data collected from time of polar body extrusion to time of starting blastulation, and trophectoderm and inner-cell-mass static morphology in all embryos cultured in a specific time-lapse incubator with a continuous medium. Logistic regressions conducted to outline the variables showing a statistically significant association with live birth. In the validation phase, these variables were tested in an independent data set. Live births per SET. The average live birth rate (LBR) in the training set was 40% (N = 207/511). Only time of morulation (tM) and trophectoderm quality were outlined as putative predictors of live birth at two IVF centers. In the validation set, the euploid blastocysts characterized by tM <80hours and high-quality trophectoderm resulted in a LBR of 55.2% (n = 37/67), while those with tM ≥ 80hours and a low-quality trophectoderm resulted in a LBR of 25.5% (N = 13/51). Time of morulation and trophectoderm quality are better predictors of a euploid blastocyst's reproductive competence. Our evidence was reproducible across different centers under specific culture conditions. These data support the crucial role of morulation for embryo development, a stage that involves massive morphologic, cellular, and molecular changes and deserves more investigation.

Timing of polar body extrusion is associated with body mass index, a pilot study using time lapse video microscopy

To determine timing of oocyte maturation by observing polar body extrusion in a closed time lapse incubator system, as well as to correlate the observed times to patient derived factors. Retrospective Analysis. 21 patients (57 oocytes) undergoing either elective oocyte cryopreservation (OC) or intra-cytoplasmic sperm injection (ICSI) at our center were included in this study. Patients that had not signed research consent or if no immature oocytes were available after retrieval were excluded. 20 patients underwent gonadotropin/antagonist cycles and one patient underwent estrogen priming. Patients were triggered as per our protocol when the lead follicles were 18-19 mm and all follicles were retrieved at the time of egg retrieval. GV oocytes were then placed in a time lapse/stage top incubator which maintains constant temp of 37 degrees C and 5% C02 in air and 95% humidity levels as a conventional incubator. Images were obtained every 5 minutes for a total of 48 hours. All times were normalized to time of hcg trigger. Oocytes were left in Global for fertilization (Lifeglobal) media (G2), which is the media the oocytes were placed in after retrieval. Average age of all participants was 38.1 +/-3.4 years. Average baseline estradiol (E2) and follicle stimulating hormone (FSH) were 49.3 +/-14.6 (pg/ml) and 6.4+/-2 (mIU/ml) respectively. Average gonadotropin dosage (IU), E2 at trigger and number of oocytes retrieved were 3778.6 +/- 1576, 3349.3 +/-1334.7 and 21.9 +/-12.6, respectively. AMH and BMI were 3.34 +/- 2.1 and 20.1 +/- 8.5 respectively. Average time to oocyte polar body (PB) extrusion (in relation to trigger time) was 3495.3 +/- 253.9 minutes. Oocytes matured in 1104.6 +/- 274.5 minutes on average once placed in the time lapse incubator. 34/57 (60%) GV oocytes matured to metaphase 2 in vitro. Multiple linear regression revealed that the time to polar body extrusion was associated with BMI but not with AMH or percent of retrieved oocytes that were mature. Time to PB extrusion (TPBe) declined with BMI according to the equation: TPBe = -35.031 (BMI) + 4290.8 (minutes). Maturation rates of 60% were seen with the above protocol, which is comparable to published rates of IVM specific media. Although age and AMH did not show significance, higher BMI did show shorter maturation rates of GV oocytes in vitro. Further studies are needed to confirm these findings.

Novel Ca2+ increases in the maturing oocytes of starfish during the germinal vesicle breakdown

It has been known that the intracellular Ca2+ level transiently rises at the specific stages of mitosis such as the moment of nuclear envelope breakdown and at the metaphase–anaphase transition. Comparable intracellular Ca2+ increases may also take place during meiosis, as was intermittently reported in mouse, Xenopus, and starfish oocytes. In a majority of starfish species, the maturing oocytes display an intracellular Ca2+ increase within few minutes after the addition of the maturation hormone, 1-methyladenine (1-MA). Although starfish oocytes at meiosis also manifest a Ca2+ increase at the time of polar body extrusion, a similar Ca2+ increase has never been observed during the envelope breakdown of the nucleus (germinal vesicle, GV). Here, we report, for the first time, the existence of an additional Ca2+ response in the maturing oocytes of Asterina pectinifera at the time of GV breakdown. In contrast to the immediate early Ca2+ response to 1-MA, which is independent of external Ca2+ and takes a form of intracellular Ca2+ wave traveling three times as fast as that in the fertilized eggs, this late stage Ca2+ response comprised a train of numerous spikes representing Ca2+ influx. These Ca2+ spikes coinciding with GV breakdown were mostly eliminated when the GV was removed from the oocytes prior to the addition of 1-MA, suggesting that the Ca2+ spikes are rather a consequence of the GV breakdown. In support of the idea that these Ca2+ spikes play a physiological role, the oocytes matured in calcium-free seawater had a higher rate of cleavage failure 2h after the fertilization in natural seawater. Specific inhibitors of L-type Ca2+ channels, verapamil and diltiazem, severely suppressed the amplitude of the individual Ca2+ spikes, but not their frequencies. On the other hand, latrunculin-A (LAT-A), which promotes net depolymerization of the actin cytoskeleton, had a dual effect on this late Ca2+ response. When added immediately after the hormone-dependent period, LAT-A inhibited the occurrence (frequency) of the spikes in a dose-dependent manner, but the amplitude of the prevailing Ca2+ spikes itself was rather significantly increased. These results suggest that the cortical actin cytoskeleton and some nuclear factors may play a role in regulating ion channel activities during this stage of meiotic progression.

The Aurora kinase inhibitor ZM447439 accelerates first meiosis in mouse oocytes by overriding the spindle assembly checkpoint

Previous studies have established that when maturing mouse oocytes are continuously incubated with the Aurora inhibitor ZM447439, meiotic maturation is blocked. In this study, we observe that by altering the time of addition of the inhibitor, oocyte maturation can actually be accelerated by 1 h as measured by the timing of polar body extrusion. ZM447439 also had the ability to overcome a spindle assembly checkpoint (SAC) arrest caused by nocodazole and so rescue polar body extrusion. Consistent with the ability of the SAC to inhibit cyclin B1 degradation by blocking activation of the anaphase-promoting complex, we could also observe a rescue in cyclin B1 degradation when ZM447439 was added to nocodazole-treated oocytes. The acceleration of the first meiotic division by ZM447439, which has not been achieved previously, and its effects on the SAC are all consistent with the proposed mitotic role of Aurora B in activating the SAC. We hypothesize that Aurora kinase activity controls the SAC in meiosis I, despite differences to the mitotic cell cycle division in spindle architecture brought about by the meiotic mono-orientation of sister kinetochores.

Successful delivery after the transfer of embryos obtained from a cohort of incompletely in vivo matured oocytes at retrieval time

To report a case of successful delivery of a healthy baby after intracytoplasmic sperm injection (ICSI) in a patient with no mature oocytes at the time of oocyte retrieval. Case report. Department of Reproductive Medicine. After a controlled ovarian hyperstimulation cycle, a total of seven immature oocytes were collected. Medical management. The timing of polar body extrusion was checked every 2 hours, and ICSI was performed as soon as the first polar body was extruded. Following incubation in culture medium, five oocytes reached the metaphase II stage within 8-8.5 hours. Three oocytes were fertilized after ICSI, and two of three cleaved embryos were transferred on day 3. The embryo transfer was followed by a single pregnancy and the delivery of a healthy baby. This case report demonstrates that embryos obtained from in vitro matured oocytes retain the developmental competence for full-term. It illustrates the importance of regularly monitoring the polar body extrusion when all collected oocytes are immature.

Timed IVM followed by ICSI in a patient with immature ovarian oocytes

A complete failure of meiotic maturation occasionally occurs following human chorionic gonadotrophin administration during IVF—intracytoplasmic sperm injection (ICSI) cycles. ICSI on day 1 is commonly used to allow maturation in culture. However, if the oocytes become mature in the evening soon after their recovery but ICSI is delayed until the next day, then subsequent ageing of matured oocytes may be unfavourable for fertilization and development. To avoid the deterioration associated with oocyte ageing, the timing of polar body extrusion was checked every 3 h and rescue in-vitro maturation (IVM)— ICSI was performed shortly after the polar body extrusion was confirmed. This report describes a successful pregnancy and birth of a healthy baby in a patient who had no mature oocytes at the time of oocyte retrieval, and illustrates the value of extra monitoring for IVM and ICSI in cases where only immature oocytes are available.

Calmodulin-dependent protein kinase II, and not protein kinase C, is sufficient for triggering cell-cycle resumption in mammalian eggs

Mouse eggs arrest at metaphase II following ovulation and are only triggered to complete meiosis when fertilized. Sperm break the cell-cycle arrest by a long-lasting series of Ca2+ spikes that lead to an activation of the anaphase-promoting complex/cyclosome. The signal transduction pathway is not fully resolved but both protein kinase C (PKC) and calmodulin-dependent protein kinase II (CamKII) activities increase at fertilization and previous pharmacological studies have implicated both in cell-cycle resumption. We have used a combination of pharmacological inhibitors and constitutively active cRNA constructs of PKCalpha and CamKIIalpha microinjected into mouse eggs to show that it is CamKII and not PKC that is the sufficient trigger for cell-cycle resumption from metaphase II arrest. Constitutively active PKC constructs had no effect on the resumption of meiosis but caused an immediate and persistent elevation in intracellular Ca2+ when store-operated Ca2+ entry was stimulated. With respect to resumption of meiosis, the effects of constitutively active CamKII on eggs were the same as sperm. Eggs underwent second polar body extrusion and pronucleus formation with normal timings; while both securin and cyclin B1 destruction, visualised by coupling to fluorescent protein tags, were complete by the time of polar body extrusion. Induction of a spindle checkpoint by overexpression of Mad2 or by spindle poisons blocked CamKII-induced resumption of meiosis, but the Ca2+ chelator BAPTA did not. Furthermore direct measurement of Ca2+ levels showed that CamKII did not induce exit from metaphase II arrest by raising Ca2+. Therefore, we conclude that PKCs may play an important role in maintaining Ca2+ spiking at fertilization by promoting store-operated Ca2+ entry, while CamKII transduces cell-cycle resumption, and lies downstream of sperm-induced Ca2+ release but upstream of a spindle checkpoint. These data, combined with the knowledge that CamKII activity increase at fertilization, suggest that mouse eggs undergo cell-cycle resumption through stimulation of CamKII.

290. Calmodulin-dependent protein kinase II, and not protein kinase C, transduces the Ca2+ signal at fertilization

Mouse eggs arrest at metaphase II following ovulation and are only triggered to complete meiosis when fertilized. Sperm break the cell cycle arrest by a long-lasting series of Ca2+ spikes that lead to an activation of the anaphase-promoting complex. The signal transduction pathway is not fully resolved but both protein kinase C (PKC) and calmodulin-dependent protein kinase II (CamKII) activities increase at fertilization and previous pharmacological studies have implicated both in cell cycle resumption. Here we used a combination of pharmacological inhibitors and constitutively-active cRNA constructs of PKCα and CamKII microinjected into mouse eggs, to show that it is CamKII and not PKC that is the sufficient trigger for cell cycle resumption from metaphase II arrest. Constitutively active PKC constructs had no effect on meiotic resumption but caused an immediate and persistent elevation in intracellular Ca2+ when store-operated Ca2+ entry was stimulated. With respect to meiotic resumption, the effects of constitutively-active CamKII on eggs were the same as sperm. Eggs underwent second polar body extrusion and pronucleus formation with normal timings; while both securin and cyclin B1 destruction, visualised by coupling to fluorescent protein tags, were complete by the time of polar body extrusion. Induction of a spindle checkpoint by overexpression of Mad2 or by spindle poisons blocked CamKII-induced meiotic resumption but the Ca2+ chelator BAPTA did not. Furthermore direct measurement of Ca2+ levels showed that CamKII did not induce exit from metaphase II arrest by raising Ca2+. Therefore we conclude that PKCs may play an important role in maintaining Ca2+ spiking at fertilization by promoting store-operated Ca2+ entry, while CamKII transduces cell cycle resumption, and lies downstream of sperm-induced Ca2+ release but upstream of a spindle checkpoint. These data, combined with the knowledge that CamKII activity increase at fertilization, suggest that mouse eggs undergo cell cycle resumption through stimulation of CamKII.

301EFFECT OF THE MII-AGE AND ACTIVATION PROTOCOL ON THE PARTHENOGENETIC DEVELOPMENT OF PORCINE OOCYTES

The completion of porcine oocyte nuclear maturation (MII) in vitro, characterized by the time of polar body extrusion, starts at about 32h of maturation and lasts more than 12h. This leads to the simultaneous presence in the population of matured oocytes with differing abilities to be activated. We investigated age-dependent changes in pig oocyte maturation, activation and development in SOFaa in response to electric impulse (EL) in the presence of cytochalasin B (CB) and EL in combination with cycloheximide and cytochalasin B (EL+CHX+CB). Oocytes were matured in TCM 199 with 10% FCS, cysteine, LH, FSH (Pergovet, Serono, Geneva, Switzerland) for 36h and then decumulated. Matured oocytes were activated at 40 and 44h by double pulse of 30μs DC 1, 5kVcm−1 and cultured in 5μgmL−1 CB for 4h or by EL followed by incubation in 10μgmL−1 CHX+5μgmL−1 CB for 4h. According to the MII-age before activation oocytes were divided into 2 age classes: 3–7 and 7–11h after polar body extrusion. Embryos were cultured in SOFaa in 5% CO2, 5% O2 at 38.5°C. The rates of cleavage, blastocyst formation and cell number of BL on Day 7 (BLD7) were recorded. Our results showed that the average rate of maturation at 44h was 72% (n=1377). About 50% and 87% of oocytes, that eventually matured, extruded the polar body at 37 and 40h, respectively. The average cell number of BLD7 developed in SOFaa was 80±36 (n=52) and was not affected by activation protocol. Seventy-nine and 27% of BL had more than 50 and 100 cells per BL, respectively. Porcine oocytes activated by EL acquired their developmental competence gradually, achieving the highest rates of cleavage and blastocyst formation 7h after polar body extrusion. By contrast, oocytes activated by EL+CHX+CB showed their maximal developmental competence earlier (3–7h group). In conclusion, we demonstrate that electric impulse in combination with CHX+CB treatment permits earlier efficient activation of porcine oocytes (3–7h after polar body extrusion).

Ca2+ oscillations at fertilization in mammals are regulated by the formation of pronuclei.

In mammals, the sperm triggers a series of cytosolic Ca(2+) oscillations that continue for approximately 4 hours, stopping close to the time of pronucleus formation. Ca(2+) transients are also seen in fertilized embryos during the first mitotic division. The mechanism that controls this pattern of sperm-induced Ca(2+) signalling is not known. Previous studies suggest two possible mechanisms: first, regulation of Ca(2+) oscillations by M-phase kinases; and second, regulation by the presence or absence of an intact nucleus. We describe experiments in mouse oocytes that differentiate between these mechanisms. We find that Ca(2+) oscillations continue after Cdk1-cyclin B1 activity falls at the time of polar body extrusion and after MAP kinase has been inhibited with UO126. This suggests that M-phase kinases are not necessary for continued Ca(2+) oscillations. A role for pronucleus formation in regulating Ca(2+) signalling is demonstrated in experiments where pronucleus formation is inhibited by microinjection of a lectin, WGA, without affecting the normal inactivation of the M-phase kinases. In oocytes with no pronuclei but with low M-phase kinase activity, sperm-induced Ca(2+) oscillations persist for nearly 10 hours. Furthermore, a dominant negative importin beta that inhibits nuclear transport, also prevents pronucleus formation and causes Ca(2+) oscillations that continue for nearly 12 hours. During mitosis, fluorescent tracers that mark nuclear envelope breakdown and the subsequent reformation of nuclei in the newly formed two-cell embryo establish that Ca(2+) oscillations are generated only in the absence of a patent nuclear membrane. We conclude by suggesting a model where nuclear sequestration and release of a Ca(2+)-releasing activity contributes to the temporal organization of Ca(2+) transients in meiosis and mitosis in mice.

Oocyte-induced haploidization

This paper describes the technical approach to treatment of age-related oocyte aneuploidy. Although one solution can be oocyte/embryo selection, another is represented by the nuclear transplantation procedure. The efficiency of nuclear transplantation into immature oocytes is described as a way of generating embryos, and the possibility that viable female gametes can be constructed by transfer of diploid somatic cell nuclei into enucleated oocytes. Germinal vesicle (GV)-stage mouse oocytes were collected from unstimulated ovaries and somatic nuclei were obtained from mouse cumulus cells obtained after ovarian stimulation. Spare human GV-stage oocytes were donated from consenting patients undergoing intracytoplasmic sperm injection (ICSI) treatment, and human somatic cells were stromal cells coming from uterine biopsies performed on consenting patients undergoing endometrial cell co-culture. GV ooplasts, prepared by enucleation, were transplanted with either GV or somatic nuclei by micromanipulation. Grafted oocytes were electrofused and cultured to allow maturation, following which they were selected at random for insemination or cytogenetic analysis. GV transplantation was accomplished with an overall efficiency of ˜80 and 70% in the mouse and the human respectively. The maturation rate of 96% (mouse) and 62% (human) following reconstitution was comparable to that of control oocytes, as was the incidence of aneuploidy among the reconstituted oocytes. The reconstituted human oocytes were successfully fertilized by ICSI at a rate of 52%. After the transfer of mouse cumulus or human endometrial cell nuclei into enucleated immature oocytes, a polar body was extruded in >40%. In a limited number of observations where the nucleus of an aged oocyte was transferred into a younger ooplasm, the chromosomes segregated normally at the time of polar body extrusion. The technique of nuclear transplantation itself did not increase the incidence of chromosomal anomalies in the mouse or human, since their oocytes reconstituted with homologous donor GV resumed meiosis to metaphase II and maintained a normal ploidy. In addition, immature mouse ooplasts induced haploidization of transplanted somatic cell nuclei. Although further evaluation of their genetic status is needed, the procedure may offer a realistic way of producing normal oocytes in cases of aged-related infertility. While the procedure is technically similar to cloning, it would generate a unique individual as a result of the contribution of both parental genomes.

Regulation of human and mouse oocyte maturation in vitro with 6-dimethylaminopurine.

It has been postulated that premature shortening of the oocyte growth phase due to the recovery of oocytes from small diameter follicles may be responsible for the developmental anomalies associated with in-vitro maturation. 6-Dimethylaminopurine (DMAP) was used to artificially lengthen the pre-maturation period of oocyte growth, in vitro, by inhibiting germinal vesicle breakdown in mouse and human oocytes. DMAP inhibited the meiotic maturation of mouse and human oocytes and the inhibition was fully reversible. The timing of polar body extrusion was accelerated in mouse oocytes following the withdrawal of DMAP; however, the kinetics of nuclear maturation in human oocytes was unaffected by exposure to DMAP. All mouse and human DMAP-treated oocytes that matured to metaphase II expressed histone H1 kinase activity. Fertilization rates in both DMAP-treated and control mouse and human oocytes were comparable, and human embryonic development was similar in control and DMAP-treated oocytes. However, blastocyst development was significantly reduced in DMAP-treated mouse oocytes (P < 0.05). It is concluded that lengthening the prematuration growth phase, by temporarily inhibiting kinase activity with DMAP, does not directly improve oocyte developmental competence but provides a useful tool for further investigating meiotic and developmentally related events in vitro by manipulating meiotic resumption.

Kinetochore fibers are not involved in the formation of the first meiotic spindle in mouse oocytes, but control the exit from the first meiotic M phase.

During meiosis, two successive divisions occur without any intermediate S phase to produce haploid gametes. The first meiotic division is unique in that homologous chromosomes are segregated while the cohesion between sister chromatids is maintained, resulting in a reductional division. Moreover, the duration of the first meiotic M phase is usually prolonged when compared with mitotic M phases lasting 8 h in mouse oocytes.We investigated the spindle assembly pathway and its role in the progression of the first meiotic M phase in mouse oocytes. During the first 4 h, a bipolar spindle forms and the chromosomes congress near the equatorial plane of the spindle without stable kinetochore- microtubule end interactions. This late prometaphase spindle is then maintained for 4 h with chromosomes oscillating in the central region of the spindle. The kinetochore-microtubule end interactions are set up at the end of the first meiotic M phase (8 h after entry into M phase). This event allows the final alignment of the chromosomes and exit from metaphase. The continuous presence of the prometaphase spindle is not required for progression of the first meiotic M phase. Finally, the ability of kinetochores to interact with microtubules is acquired at the end of the first meiotic M phase and determines the timing of polar body extrusion.

A soluble extract from human spermatozoa activates ascidian oocytes.

A soluble extract from human spermatozoa induced calcium oscillations and extrusion of the first polar body when injected into oocytes of the ascidian Ciona intestinalis. The properties of calcium oscillations and time of polar body extrusion precisely mimic oocyte activation induced by C. intestinalis sperm or sperm extracts. The data suggest that human sperm extracts can activate oocytes of different phyla by the same mechanism as homologous spermatozoa. Injection of inositol 1,4,5-trisphosphate (IP3) into C. intestinalis oocytes mimicked to some extent the initial stages of oocyte activation, but the results demonstrate that ascidian oocyte activation by human sperm extract cannot be explained solely in terms of IP3-induced calcium release. Injection of other calcium releasing second messengers, cyclic adenosine diphosphate ribose, or calcium ions, does not lead to oocyte activation or release intracellular calcium in ascidian oocyte. It was concluded that human spermatozoa contain one or more molecules than can trigger intracellular calcium release in oocytes from different phyla.

Cell cycle modification during the transitions between meiotic M-phases in mouse oocytes.

When metaphase II-arrested mouse oocytes (M II) are activated very soon after ovulation, they respond abortively by second polar body extrusion followed by another metaphase arrest (metaphase III, M III; Kubiak, 1989). The M II/M III transition resembles the natural transition between the first and second meiotic metaphases (M I/M II). We observed that a similar sequence of events takes place during these two transitions: after anaphase, a polar body is extruded, the microtubules of the midbody disappear rapidly and a new metaphase spindle forms. The MPM-2 monoclonal antibody (which reacts with phosphorylated proteins associated with the centrosome during M-phase) stains discrete foci of peri-centriolar material only in metaphase arrested oocytes; during both transitional periods, a diffuse staining is observed, suggesting that these centrosomal proteins are dephosphorylated, as in a normal interphase. However, the chromosomes always remain condensed and an interphase network of microtubules is never observed during the transitional periods. Incorporation of 32P into proteins increases specifically during the transitional periods. Pulse-chase experiments, after labeling of the oocytes in M phase with 32P, showed that a 62 kDa phosphoprotein band disappears at the time of polar body extrusion. Histone H1 kinase activity (which reflects the activity of the maturation promoting factor) drops during both transitional periods to the level characteristic of interphase and then increases when the new spindle forms. Both the M I/M II and M II/M III transitions require protein synthesis as demonstrated by the effect of puromycin. These results suggest that the two M-phase/M-phase transitions are probably driven by the same molecular mechanism.