Normal Cleavage Pattern Research Articles

P-218 Should embryos with direct unequal cleavage be given a chance or directly deprioritized? A follow-up on blastocyst formation and ploidy

Abstract Study question Should embryos with direct unequal cleavage (DUC) be given a chance to develop to blastocyst for a quality and ploidy assessment or directly deprioritized? Summary answer Although DUC embryos are clearly associated with poorer outcomes, if DUC embryos develop to good quality blastocysts the euploidy rate is comparable to non-DUC embryos. What is known already DUCs were first defined by time-lapse imaging and their implantation rate was found to be significantly lower than embryos with a normal cleavage pattern (1.2% vs. 20.2%, respectively) leading to the suggestion that rejection of these embryos for transfer could improve implantation rates (Rubio et al., 2012). Another study concluded that blastocyst formation, implantation potential and euploidy rate are significantly reduced in DUC embryos and that they should be deselected for day 3 transfers but should be cultured to blastocyst stage for possible embryo transfer (Zhan et al., 2016). However, in practice, DUCs are deselected and are therefore rarely transferred. Study design, size, duration This retrospective study included 1191 DUC embryos and 5058 non-DUC embryos incubated in the Embryoscope in 2022. Twenty-eight DUC and 185 non-DUC embryos were transferred in a fresh cycle. Preimplantation genetic testing for aneuploidy (PGT-A) was performed on 128 DUC and 1477 non-DUC blastocysts. Five DUC embryos were transferred in a frozen-thawed cycle. Participants/materials, setting, methods A total of 2430 IVF patients treated in a single private clinic were included in this retrospective study. The mean female age of the cohort was 36.5. Some of the blastocysts were subjected to PGT-A mainly for advanced maternal age. CHLOE (Fairtility) software analyzed time-lapse videos and identified pronuclei, DUCs and blastulation. DUC embryo outcomes (ploidy, blastulation and blastocyst quality) were assessed. Direct cleavages from 2 to 5 cells were not included in the study. Main results and the role of chance The outcomes of DUC embryos (n = 1191) were compared to non-DUC embryos (n = 5058). The blastulation rate was 23.8% for DUC embryos and 50.2% for non-DUC embryos. The good-quality blastocyst rate was 15.1% and 42.5%, respectively. The top-quality blastocyst rate was 2.4% and 18.5%, respectively. PGT-A was performed for 128 DUC blastocysts; 52 were euploid (40%) and 14 were mosaic (11%). The euploidy rate for non-DUC blastocysts was 43% and the mosaicism rate was 9.4%. Thirty-three DUC embryos were transferred giving 6 live births (18%). Limitations, reasons for caution The discrimination between fragments and blastomeres within the 5 hours from the first division is challenging for AI algorithms. Therefore, some DUC embryos may have been misclassified. Wider implications of the findings In conclusion, although DUC embryos are not prioritized for transfer, our results show that, once achieving the blastocyst stage with a good quality, their euploidy rate is comparable to non-DUC blastocysts. Morphokinetic analysis may propose additional markers to identify higher implantation potential DUC blastocysts. Trial registration number N/A

P-204 The pattern of the zygote cleavage to three cells must be considered before embryo transfer

Abstract Study question What is the clinical outcome of instant irregular cleavage (IDC) from zygote to three cells? Summary answer IDC embryos, did not develop to blastocysts and did not acieve pregnancy; FC embryos that reached blastocyst stage exerted similar pregnancy rates as control embryos What is known already Evaluation of the zygote morphology is one of the earliest embryonic stages investigated. Several zygote grading systems were proposed based on pronuclei size and NPB (nucleolus precursor bodies) distribution, thus is an effective indicator of the embryo’s potential to result in a life birth. The first cleavage of a zygote is a highly coordinated event. Disruptive timing of the first cleavage, named direct cleavage (DC, less than 5 hours), is associated with formation of poor embryo quality. Early identification of pathological embryos is challenging and impacts the timing of embryo fate decision in ART. Study design, size, duration A total of 1978 fresh IVF cycles from single unit were analyzed and a total of 4012 embryos were studied. Only 2pn embryos, cultured exclusively in time-lapse system for 5 days were included. The morphokinetics of embryos and their outcome were recorded, from z-score analysis through day 5. We discriminated between IDC, FC and normal cleavage pattern of embryos and compared clinical outcome of various morphokinetic parameters. We also evaluated possible early predictors for IDC. Participants/materials, setting, methods We generated three study groups according to embryo cleavage pattern: (I) Control, normal cleavage (n = 551); (II) FC, zygote to three cells within 5 hours (n = 1587); (III) IDC, instant cleavage from zygote to three cells (n = 922). The association between z-score of 138 IDC embryos and their sibling random controls was thoroughly investigated. All time lapse annotations were performed by senior embryologists. Statistical analysis was performed by SPSS software. Main results and the role of chance IDC embryos were mainly arrested at day 3 and the number of usable embryos (reached blastocyst stage and were suitable for embryo transfer or cryopreservation) was negligible; 4/922 (0.4%). In comparison, the amount of usable FC embryos was 108/1587 (6.6%) and control embryos usage reached 180/551 (32.7%). While the pregnancy rate of control and FC embryos, which reached embryo transfer were similar (40.35% and 42.55%, respectively); transfer of IDC embryos did not result in pregnancy. Additionally, the timetable as measured by time of PN fading and time from fading to first cleavage, differed significantly between the three groups. Therefore, we performed a thorough analysis of zygote morphology data of IDC embryos compared to control sibling embryos in search of early possible markers for these findings. We have not detected significant differences in z-score analysis, reflected by number and size of nucleoli, as well as pronuclear symmetry. Limitations, reasons for caution IDC embryos number used for z-score analysis was limited since many didn’t meet the inclusion criteria (lack of overlapping of the pronuclei, presence of random of control embryo and good quality image). Wider implications of the findings The decision regarding the fate of IDC and FC embryos should include the pattern of first cycle cleavage. Culture IDC and FC embryos for 5 days up to the blastocyst will spare transfer of embryos that are fated to arrest even when their morphological grade on day 3 is acceptable. Trial registration number 0043-22-MMC

O-219 Detailed morpho-kinetic analysis of the first cleavage can help in evaluating the viability of direct-cleaved human zygotes

Abstract Study question Why do some direct-cleaved human zygotes still lead to a live birth? Summary answer Direct-cleaved zygotes which have undergone the 2-cell stage can lead to a live birth, while zygotes cleaved from 1-cell to ≥ 3-cell do not. What is known already In recent years, zygotes that develop from 2-cell to 3-cell within 5 hours after the first cleavage have been evaluated as “direct-cleaved” zygotes, because normal cleavage takes approximately 12 hours to complete. It was reported that their implantation rate was significantly lower than zygotes with normal cleavage pattern, and eliminating direct-cleaved zygotes from transfer could improve the implantation rate. However, some direct-cleaved zygotes at the first cleavage could still lead to a live birth. Few reports have examined the difference between a cleavage from 1-cell to ≥ 3-cell and 2-cell to ≥ 3-cell within 5 hours after the first cleavage. Study design, size, duration A retrospective study involving 2,077 cycles of IVF/ICSI between July 2012 and July 2019. A total of 5,991 normally fertilized zygotes (2PN/2PB) were included. Of those, 3,508 were evaluated as usable good/fair quality embryos on Day2/3, and the rest (n = 2,483) were evaluated as poor quality and rejected from transfer or cryopreservation after 7 days of culture. Of 3,508 usable embryos, 884 were selected based on the availability of results of live birth for this study. Participants/materials, setting, methods Time-lapse imaging (5 slices along Z-axis every 10 minutes) was performed in EmbryoScopeTM. Zygotes were morphokinetically analyzed in detail and classified into four groups by their cleavage patterns: Group1 (1-cell→2-cell); Group 2 (1-cell→3-cell); Group 3 (1-cell→2-cell→≥3-cell within 5 hours after the first cleavage); and Group 4 (1-cell→2-cell→≥5-cell). The proportion, mean maternal age and live birth rate of each group were examined. Main results and the role of chance The proportion of Groups 1-4 was 83.6% (n = 739), 3.8% (n = 34), 5.9% (n = 52), and 6.7% (n = 59), respectively. 0f 884 zygotes examined in this study, the mean maternal age was significantly higher in Group 2 and 4 than in Group 1 (P < 0.05; 37.4±4.9 in Group1, 39.1±5.2 in Group 2, 38.6±6.0 in Group 3, and 38.7±5.1 in Group 4). The rate of confirmed gestational sac was significantly lower in Group 2 and 4 than in Group 1 [P < 0.01; 36.3% (n = 268/739), 0% (n = 0/34), 25.0% (n = 13/52), and 18.6% (n = 11/59) in Groups 1-4, respectively]. Furthermore, the live birth rate was significantly higher in Group 1 than in Groups 2, 3 and 4 [P < 0.01; 28.4% (n = 210/739), 0% (n = 0/34), 13.5% (n = 7/52), and 15.3% (n = 9/59) in Groups 1-4, respectively]. Above all, while zygotes in Group 2 showed no pregnancy and live birth at all, zygotes in Group 3 showed a live birth rate of 13.5%. However, they had a significantly higher miscarriage rate (42.9%, n = 6) compared to zygotes in Group 1 (19.5%, n = 55). Limitations, reasons for caution It is very difficult to capture cleavage patterns by routine observations because the timings of developmental events are different between embryos. A time-lapse imaging and culturing system is essential to solve this problem, however, it cannot visualize the distribution of chromosomes, and no chromosomal analysis was conducted in this study. Wider implications of the findings This study revealed that zygotes previously classified as “direct-cleaved” and eliminated from transfer included viable zygotes which could lead to a live birth. Therefore, it is crucial to optimize the use of time-lapse imaging of human zygotes in order to precisely evaluate the first cleavage. Trial registration number not applicable

179 RELATIONSHIP BETWEEN GENE EXPRESSION IN INDIVIDUAL BLASTOMERES OF 2-CELL STAGE BOVINE EMBRYOS AND THE NORMALITY OF FIRST CLEAVAGE

Previously early first and second cleavages after IVF associated with even blastomeres without fragments or protrusions were found to be a potent criterion for the selection of embryos with high development competence (Sugimura et al. 2012 PLOS One 7, e36627). The aim of this study was to examine the relationship between an early normal first cleavage pattern and the transcript abundance in each blastomere in 2-cell stage bovine embryos. IVF-derived bovine embryos were cultured individually in microwells culture dish in CR1aa medium supplemented with 5% calf serum and 0.25 mg mL–1 linoleic acid albumin at 38.5°C in 5% CO2, 5% O2, and 90% N2. First cleavage and cleavage patterns were categorised as being either normal (the first cleavage within 28 h after IVF with 2 even blastomeres without fragment or protrusion) or abnormal (2 uneven blastomeres, with/without fragment/protrusion and/or later than 28 h after IVF at the first cleavage). Next, cleaved embryos were placed in 0.5% actinase-E in Ca- and Mg-free PBS and blastomeres were separated by pipetting. Individual blastomeres (n = 71, 10 replicates) were analysed for gene expression by quantitative RT-PCR. Primers were designed for 19 target genes related to pluripotency, cell cycle, metabolism, pregnancy reorganization, placentation and fetal growth (NANOG, OCT4, PLAC8, ATP1A1, CCNB1801, CDH1, COX1, CTNNB1, G6PDH, Glut8, MNSOD-3end, SOX2, DYNLL1, IGF1R, IGF2, IGF2R, IGFBP2, IGFBP3, and PMSB1) and a reference gene (PPIA). Transcript abundance of target genes in both of individual blastomeres of cleaved embryos was examined in embryos that cleaved early with a normal cleavage pattern and in those that showed abnormal cleavage pattern. Values were normalised to the average values of the reference genes and means were compared by the student t-test. Transcript abundance of OCT4, ATP1A1, CCNB1801, CDH1, COX1, CTNNB1, MNSOD-3end, IGF2R, and IGFBP2 was significantly higher in blastomeres associated with all categorised abnormal blastomeres compared towith an early normal cleavage (P < 0.05). Furthermore, the expression of PLAC8, IGF1R, and PMSB1 in embryos having 2 uneven blastomeres, Glut8 and SOX2 in 2 uneven blastomeres with fragment/protrusion was higher than that in normal cleavage (P < 0.05). However, the level of G6PDH was lower in embryos having 2 uneven blastomeres than that in those showing normal cleavage (P < 0.05). Our results reveal blastomere gene expression in bovine embryos at the first cleavage may correlated with oocyte developmental competence. This study was supported by JSPS KAKENHI (26450388).

237 THE EFFECTS OF BULLS AND X-SORTING OF SPERM ON THE ACCURACY OF NONINVASIVE CRITERIA TO PREDICT BLASTOCYST FORMATION OF IN VITRO-PRODUCED BOVINE EMBRYOS

Previously, an early first cleavage and a second cleavage after IVF with a normal cleavage pattern defined by even blastomeres without fragments or protrusions was found to be a potent marker for the selection of embryos with high developmental competence (Sugimura et al. 2012 PLoS ONE 7, e36627). The aim of this study was to investigate the effects of bulls and X-sorting of sperm on the ability of these simple noninvasive markers to predict the potency of bovine IVF embryos to develop to the blastocyst stage in vitro. Immature oocytes were matured in TCM199 supplemented with 0.02 armour unit mL–1 FSH and 5% calf serum at 38.5°C in 5% CO2 and 95% air for 22 to 23 h. After maturation, oocytes were inseminated with either of non-sorted frozen-thawed sperm from 3 bulls (A–C) or X-sorted sperm of bull A. Putative zygotes were cultured (IVC) in CR1aa medium supplemented with 5% calf serum and 0.25 mg mL–1 linoleic acid albumin at 38.5°C in 5% CO2, 5% O2, and 90% N2 for 216 h. Embryo kinetics were observed individually by time-lapse cinematography (CCM-1.3Z; Astec, Fukuoka, Japan; Sugimura et al. 2010 Biol. Reprod. 83, 970–978). First and second cleavage kinetics and pattern were categorized according to Sugimura et al. (2012). For each bull, blastocyst development from embryos possessing the following 3 selection markers was compared: (marker 1) the first cleavage within 28 h after IVF, (marker 2) marker 1 combined with 2 even blastomeres without fragments or protrusions, and (marker 3) marker 2 combined with the second cleavage within 50 h after IVF with ≥6 even blastomeres without fragments or protrusions, respectively. Data were analysed by the Yates' corrected chi-square test. A total of 823 oocytes were used in at least 3 replications. When non-sorted sperm was used for IVF, there was not difference (P > 0.05) in total blastocyst formation rates on Day 8 (Day 0 = IVF) among bulls (ranging between 49.5 and 60.8%); however, blastocyst formation rate of embryos generated from X-sorted sperm of bull A (39.5%) was lower (P < 0.05) compared with other groups despite of similar cleavage rates. Embryos having marker 3 criteria developed to the blastocysts stage at significantly higher rates than those having marker 1 criteria in case of non-sorted sperm of bulls A, B, C, and X-sorted sperm of bull A (75.9, 87.0, 90.0, and 75.0% v. 59.5, 62.2, 63.6, and 46.3%, respectively). In groups produced from non-sorted sperm of bulls A, B, C, and X-sorted sperm of bull A, blastocyst development rates of embryos with marker 2 criteria (73.7, 75.0, 90.0, and 65.8%, respectively) were higher (P < 0.05) than those of embryos having marker 1 criteria but did not differ significantly from those with marker 3 criteria. Our results reveal that a first cleavage within 28 h after IVF to 2 even blastomeres without fragments or protrusions are potent predictive markers of the developmental competence of bovine embryos to the blastocyst stage regardless of bulls and sperm sorting.Research was partly supported by JSPS KAKENHI (26450388).

236 KINETICS AND PATTERN OF THE FIRST CLEAVAGE OF IN VITRO-FERTILIZED EMBRYOS BY IN VIVO-MATURED OOCYTES AND X-SORTED SPERMATOZOA IN DAIRY CATTLE

Recently, we reported that high rates of good-quality blastocysts can be produced by IVF of in vivo-matured oocytes, obtained by ovum pick-up (OPU) after superstimulation in Holstein cows, with X-sorted sperm [Matoba et al. 2012 Reprod. Domest. Anim. 47(Suppl. 4), 515]. However, we have limited knowledge concerning the normality of embryonic cleavages in such embryos. The present study examined their kinetics and pattern of the first cell cycle. In vivo-matured oocytes were collected by OPU from non-lactating Holstein cows just before ovulation after superstimulation and ovulation induction by gonadotropin-releasing hormone. The oocytes were inseminated with 5 × 106 sperm mL–1 of X-sorted sperm and cultured in CR1aa supplemented with 5% newborn calf serum and 0.25 mg mL–1 of linoleic acid albumin at 38.5°C in 5% CO2, 5% O2, and 90% N2 for 216 h. Embryo kinetics were observed individually using a microwell culture dish and time-lapse cinematography (CCM-1.4MZS, Astec, Fukuoka, Japan) (Sugimura et al. 2010 Biol. Reprod. 83, 970–978). Photographs of each embryo were taken every 15 min during the in vitro culture period, and images were analysed by CCM-1.4 software (Astec). The cleavage pattern was categorised into normal cleavage (2 even blastomeres without fragment or protrusion) or abnormal cleavage (those with 2 uneven blastomeres, with fragments or protrusions and those dividing into 3 to 5 blastomeres at the first cleavage). Data were analysed by ANOVA, chi-square, and discriminant function. A total of 117 embryos were examined; of this number, 63.2% developed to the blastocyst stage and the rest were degenerated. A high rate of normal cleavage and a low rate of abnormal cleavage, including those with 2 uneven blastomeres and those with fragments or protrusions in the first cleavage pattern, were recorded in embryos that could develop to blastocysts compared with degenerated ones (P < 0.01 or P < 0.05, respectively; Table 1). No significant difference was found in those dividing into 3 to 5 blastomeres between the blastocysts and degenerated embryos (Table 1). Embryos developing to the blastocyst stage had a shorter duration of the first cell cycle [27.2 ± 2.3 h post-insemination (hpi)] compared with those undergoing degeneration (30.6 ± 5.7 hpi; P < 0.001). The threshold of duration of the first cell cycle was calculated by (X – 27.2)/2.3 = (30.6 – X)/5.7, resulting in X = 28.2. Blastocysts with a short duration of the first cell cleavage (≤28.2 hpi) showed a higher frequency of the normal cleavage pattern than those with a duration of the first cell cleavage longer than 28.2 hpi (71.7 and 53.6%, respectively; P < 0.05). Our results revealed that those IVF embryos that finished their first cleavage before 28.2 h of IVF and showed a normal cleavage pattern had superior developmental competence. Table 1.The first cleavege pattern reflects the developmental competence: blastocysts versus degenerated embryos This work was supported by the Research and Development Projects for Application in Promoting New Policy of Agriculture, Forestry and Fisheries (22016).

Limited implantation success of direct-cleaved human zygotes: a time-lapse study

To evaluate embryos with direct cleavage (≤5 hours) from two to three cells (DC2-3) and correlate this morphokinetic parameter to implantation and ongoing pregnancy. Clinical multicenter retrospective study. Private invitro fertilization (IVF) centers. From three clinics, a total of 979 treatments including 5,225 embryos using autologous or donated oocytes, of which 1,659 embryos were transferred. None. Clinical pregnancy confirmed by ultrasound in week7. Of the total embryo cohort, 715 (13.7%) underwent direct cleavage from two to three cells, 1,659 embryos were transferred to recipients, and 109 of the transferred embryos cleaved directly from two to three cells (6.6%). Only one DC2-3 embryo was known to result in a clinical pregnancy (1%) and 80 (73.4%) DC2-3 embryos did not implant. Of the 1,550 embryos transferred not showing DC2-3, 203 embryos were from treatments with 100% implantation (13.1%), and 804 (51.8%) embryos did not implant. The known implantation rate of DC2-3 embryos was statistically significantly lower than for embryos with a normal cleavage pattern (1.2% vs. 20.2%, respectively). Embryos with DC2-3 had a statistically significantly lower implantation rate than embryos with a normal cleavage pattern, suggesting that rejection of these embryos for transfer could improve the implantation rate.

The Diversity of the Immune Response to the A2 Domain of Human Factor VIII In a Murine Hemophilia A Model

AbstractAbstract 2223Factor VIII (fVIII) inhibitory antibodies (fVIII inhibitors) are a significant source of morbidity in patients with hemophilia A. Approximately 30% of patients with severe hemophilia A will develop inhibitors. Most inhibitors are directed against either the A2 or C2 domains of fVIII. Anti-A2 antibodies have been reported to bind to at least 3 different regions of the A2 domain. Murine anti-human fVIII monoclonal antibody (MAb) MAb413 has an epitope that localizes to amino acids 484–508 and possibly blocks factor IXa binding to fVIII. R8B12 is a minimally inhibitory anti-A2 MAb that recognizes a discontinuous epitope that includes residues 497–510 and 584–593. CLB-Cag 9 is an anti-A2 MAb that recognizes amino acids 713–740 and decreases the rate of light chain cleavage by thrombin and factor Xa.The goal of this study was to investigate the diversity of the humoral immune response to the A2 domain of human fVIII in a murine hemophilia A model. A panel of 10 murine anti-A2 antibodies was obtained, which included 9 MAbs from anti-fVIII hybridomas produced in our laboratory and antibody MAb413. All 10 MAbs were used as primary antibodies in a competition ELISA using human fVIII as the antigen. The same panel was biotinylated and used as secondary antibodies. Antibody pairs were classified as having non-overlapping or overlapping epitopes based on whether the binding of the secondary antibody was present or absent, respectively. The competition ELISA yielded 3 distinct groups of structural epitopes (see Figure). The ability of the MAbs to inhibit porcine/human hybrid fVIII proteins in a one-stage coagulation assay was used to further delineate the position of the structural epitope. Group 1 contained 6 antibodies, including MAb413, with 4 distinct epitopes on the fVIII protein. Group 2 contained 2 MAbs, and the epitope for 2–54 localized to amino acids 508–541. Group 3 contained 2 MAbs, and the epitope for 1D4 localized to amino acids 605–740.Five of the 6 MAbs in Group 1 were Type I inhibitors with titers ranging from 330-40,000 Bethesda units (BU)/mg and one was non-inhibitory (see Table). In Group 2, both antibodies had high inhibitor titers (11,000 and 33,000 BU/mg) but were type II inhibitors with residual activities of ~20% at saturating concentration of antibody. Group 2 MAb 2–54 also was tested in a purified Xase assay and the residual fVIII activity was high (60-70%) at saturating concentration. Of the two MAbs in Group 3, one was non-inhibitory and the other was a Type I inhibitor with a titer of 7000 BU/mg. The concentration dependence of MAb413 (Group 1), 2–54 (Group 2), and 1D4 (Group 3) on fVIII cleavage and activity was tested by SDS-PAGE and the intrinsic Xase assay. With MAb413 a normal cleavage pattern was seen at concentrations that produced complete inhibition in the Xase assay. With 1D4, light chain, but not heavy chain, cleavage was inhibited in a concentration-dependent manner that corresponded to a decrease in Xase activity. This is consistent with CLB-Cag 9 which has a similar epitope at the C terminal end of the A2 domain. With 2–54, cleavage at all thrombin cleavage sites was partially inhibited, which corresponded to 30–40% inhibition in the Xase assay. This MAb represents a novel class of high titer, type II anti-A2 inhibitors that recognizes residues 508–541 and has similar in vitro characteristics to the Group BC anti-C2 MAbs. The elucidation of the structural and functional complexity of the anti-fVIII A2 repertoire should be useful in the characterization of the pathogenicity of A2 inhibitors. [Display omitted] Table:A2 MAb CharacteristicsMAbInhibitor Titer (BU/mg)Inhibitor KineticsGroupStructural Epitope4A440,000Type I1aMAb41321,000Type I1b484–5082-7638,000Type I1b2-10540,000Type I1b4F4330Type I1c2-93<1n/a1d2-5433,000Type II2508–5412-10111,000Type II21D47000Type I3605–7404C7<1n/a3 Disclosures:No relevant conflicts of interest to declare.

Development of embryos from in vitro ovulated and fertilized oocytes of the quail (Coturnix coturnix japonica).

The development of quail embryos obtained after in vitro fertilization of oocytes ovulated in vitro was investigated. About 40% of the specimens, after 18-20 hr of incubation, had undergone cleavage to reach stages IV-VI when viewed under a stereo microscope. However, only 36% of these embryos contained normal, DAPI-stained nuclei when observed under a fluorescent microscope; the other 64% showing a morphologically normal cleavage pattern did not contain nuclei. Control unfertilized oocytes, ovulated in vitro and cultured for the same time, also sometimes attained the morphologically correct stages IV-VI but their "blastomeres" were always devoid of nuclei. Therefore, it is advisable to monitor early avian embryos for the presence of nuclei when assessing development in culture. The results demonstrate, for the first time, that cytoplasmic segmentation can occur in the absence of nuclear divisions in the germinal disc of the quail and show the existence and significance of ooplasmic maternal information in birds. This phenomenon is also known for sea urchin and frogs. It is indicative of the role of maternal information in early development. The in vitro method presented here links the steps of ovulation and fertilization with the early cleavage stages under in vitro conditions and may be useful in studying mechanisms of fertilization and differentiation in birds as well as in obtaining transgenic birds by DNA injection or application of foreign, DNA-carrying sperm.

A correlative study of experimentally changed first cleavage and Janus development in the trunk of Platynereis dumerilii (Annelida, Polychaeta).

Among zygotes of Platynereis dumerilii treated with cytochalasin B (CCB) prior to first cleavage, a wide variety of developmental effects were observed. One effect is a delay in the first cleavage. Treated embryos may skip the first or even more than one cleavage cycle and become multinucleated. Once these eggs start cleaving their cleavage plane takes the same position as in synchronously fertilized controls. Accordingly, the first cleavage in embryos having skipped the first normal cleavage cycle is meridional and equal, but their second cleavage is equatorial as in the third cleavage in controls. None of the embryos that were observed to skip early cleavages showed normal organogenesis, but developed into vesicle-shaped embryos with little cytological differentiation. Another effect of CCB treatment is altered blastomere size in those embryos which begin cleaving in synchrony with controls. While the majority of treated embryos followed a normal cleavage pattern, i.e. they cleaved at the right time and inequally, some of them cleaved equally or almost equally (adequally). Most of these embryos showed cleavage defects in subsequent cleavage cycles and became abnormal vesicle-shaped embryos. However, some of these embryos cleaving on schedule and equally or adequally developed into juvenile worms showing complete duplication of urites and parapodial rows (0.3% of all treated eggs) and are described as Janus duplicitates. This means that the occurrence of duplicitates and geometrically altered first cleavage patterns are correlated phenomena. The character and origin of the duplications and the consequences for dorsoventral polarity are discussed.

The role of cell lineage in development.

Studies of the role of cell lineage in development began in the latter part of the 19th century, fell into decline in the early part of the 20th, and were revived about 20 years ago. This recent revival was accompanied by the introduction of new and powerful analytical techniques. Concepts of importance for cell lineage studies include the principal division modes by which a cell may give rise to its descendant clone (proliferative, stem cell and diversifying); developmental determinacy, or indeterminacy, which refer to the degree to which the normal cleavage pattern of the early embryo and the developmental fate of its individual cells is, or is not, the same in specimen after specimen; commitment, which refers to the restriction of the developmental potential of a pluripotent embryonic cell; and equivalence group, which refers to two or more equivalently pluripotent cell clones that normally take on different fates but of which under abnormal conditions one clone can take on the fate of another. Cell lineage can be inferred to have a causative role in developmental cell fate in embryos in which induced changes in cell division patterns lead to changes in cell fate. Moreover, such a causative role of cell lineage is suggested by cases where homologous cell types characteristic of a symmetrical and longitudinally metameric body plan arise via homologous cell lineages. The developmental pathways of commitment to particular cell fates proceed according to a mixed typologic and topographic hierarchy, which appears to reflect an evolutionary compromise between maximizing the ease of ordering the spatial distribution of the determinants of commitment and minimizing the need for migration of differentially committed embryonic cells. Comparison of the developmental cell lineages in leeches and insects indicates that the early course of embryogenesis is radically different in these phyletically related taxa. This evolutionary divergence of the course of early embryogenesis appears to be attributable to an increasing prevalence of polyclonal rather than monoclonal commitment in the phylogenetic line leading from an annelid-like ancestor to insects.

Experimental Study of the Developing Mammalian Egg: Removal of the Zona Pellucida

The zona pellucida may be removed from all stages of the mouse egg by digestion with pronase. Cumulus and corona cells are also dispersed by the enzyme. No change in membrane disgestibility occurs at fertilization. In tests thus far of two-cell eggs and later stages, development continues. Blastocysts exhibit "hatching" behavior despite absence of the zona. Functions of the zona include maintenance of the normal cleavage pattern and prevention of egg fusion.