Polar Body Release Research Articles

P-264 Analysis of embryo development based on time of pronuclei appearance (tPNa) and aspects of overcoming delayed pronuclei appearance through morphokinetic patterns

Abstract Study question How much delay in the appearance of 2PN can be considered normal when predicting embryo transfer potential? Summary answer Embryos with tPNa <13hpi post-insemination develop into good-quality blastocysts; embryos with tPNa ≥13hpi develop blastocysts by shortening the time of tPNf-tPNa. What is known already Oocytes generally exhibit 2PNs at 17–20 hours, with pronuclei fading (time of pronuclei fading; tPNf) at 23–25 hours and developing into two cells (time of 2-cell division; t2) at 25–33 hours. Fertilization confirmation is typically performed 17–18 hours after insemination. However, a few oocytes with no visible PN (0PN) at the time of fertilization confirmation develop morphologically normal blastocysts, ultimately leading to pregnancy. The failure to identify PNs can be attributed to two scenarios: rapid fading or delayed appearance. Notably, there is still a lack of research on the normal range for delayed PN appearance. Study design, size, duration This study was conducted with 2153 embryos obtained from 390 intracytoplasmic sperm injection cycles (August 2021 to June 2023). All embryos were incubated for 5 days using a time-lapse system (EmbryoScopeTM+, Vitrolife). The blastocyst development rate and morphokinetic parameters according to the tPNa of embryos were analyzed using KIDScore D5 v3 and iDAScore v2.0 (VTH server+, Vitrolife). Clinical pregnancy was also analyzed. Participants/materials, setting, methods Morphokinetic parameters were analyzed from time of second polar body (tPB2) ∼ time of expanded blastocyst (tEB). The times taken for PN to appear after the second polar body release (tPNa–tPB2), for PN to fade (tPNf–tPNa) and for 2-cell division (t2–tPNf) were calculated and compared. Blastocysts were graded using the Gardner system, a grade of BB or higher divided into good quality blastocysts (GQ-BL). Main results and the role of chance tPNa was observed as 8.09±2.11hpi [1.93hpi∼32.46hpi; <5hpi (n = 49), 5∼6hpi (n = 474), 7∼8hpi (n = 1220), 9∼10hpi (n = 266), 11∼12hpi (n = 91), 13∼14hpi (n = 29), 15∼20hpi (n = 18) and >20hpi (n = 6)]. The rate of blastocysts was highest at 5∼6hpi (64.98%) and significantly lower at 9∼10hpi (54.14%), 11∼12hpi (42.86%) and 13∼14hpi (31.03%) (p < 0.005). Similarly, the rate of GQ-BL was also highest at 5∼6hpi (29.11%) and significantly lower at 9∼10hpi (19.17%), 11∼12hpi (10.99%) and 13∼14hpi (3.45%) (p < 0.005). No embryos developed into GQ-BL at 15∼20hpi, and no embryos developed into blastocysts at > 20hpi. The iDAScore was significantly different at < 13hpi and ≥13hpi (6.00±1.86 vs. 4.24±2.26, p < 0.005). Similarly, KIDScore D5 showed the same patterns (6.33±1.93 vs. 4.05±1.66, p < 0.005). At ≥ 13hpi, no blastocysts led to pregnancy. Morphokinetic parameters were analyzed to identify the factors influencing the development of blastocysts. The analysis revealed that tPNf-tPNa tended to gradually become shorter with delayed tPNa. Notably, there were significant differences in tPNf-tPNa between <13hpi and ≥13hpi (16.37±4.10 vs. 13.72±6.28, p < 0.005). At ≥ 13hpi, tPNf-tPNa was shorter in blastocysts than in cases of cleavage arrest (10.68±5.74 vs. 15.28±5.97, p < 0.05). It was particularly observed that tPNf-tPNa of GQ-BL in ≥ 13hpi was 7.90hpi, and t2 was not significantly different from blastocysts and GQ-BL in < 13hpi (25.06 vs. 25.66±3.18 vs. 25.19±2.97). Limitations, reasons for caution More data are needed for conclusive pregnancy results due to the small number of samples. Additionally, the maturation of oocytes had yet to be considered; further detailed studies related to oocyte maturity are needed. Wider implications of the findings Embryos with a tPNa of < 13hpi develop into GQ-BL. Embryos with delayed tPNa tend to overcome this by shortening the tPNf-tPNa, resulting in develop into blastocysts. Predicting blastocyst development can be achieved by considering factors such as tPNa∼t2. This can aid in improving the selection of embryos for cleavage-stage ET. Trial registration number N/A

Application of high hydrostatic pressure (HHP) shock to induce triploid development in the European grayling (Thymallus thymallus L.)

Use of parental individuals from allochthonous lineages to produce stocking specimens resulted in genetic contamination of many local European grayling (Thymallus thymallus) populations. Stocking programs including triploid individuals that are not able to reproduce with stocks located in natural habitats due to being reproductively sterile could contribute to populations of the European grayling and prevent regional lineages from the genetic pollution. The main goal of the present research, therefore, was to provide conditions for triploidization of the European grayling and examine gonads of triploid individuals. High Hydrostatic Pressure (HHP) shock (9000 psi/5 min.) applied to activated and incubated grayling eggs at 10 °C between the 17 min. 30 sec. and 20 minute timepoints subsequent to the time of insemination resulted in inhibition of the second polar body release and triploidization. Histological analysis confirmed that all 1-yr-old cytogenetically confirmed triploids had gonadal tissues indicative of sterility with the gonads being composed of connective tissue including fibroblasts, adipocytes and degenerated epithelial structures without there being any differentiated germ cells detected. There were no differences in the body length and Fulton’s condition factor between 1 year old diploid and triploid grayling, however, the average body weight was markedly less in triploid than diploid specimens. Although, 1-yr-old triploid European female and male graylings did not have fully developed gonads, before these triploid specimens are deemed safe, as a result of being reproductively sterile for stocking in natural grayling habitats, there needs to be further examination of the 2- and 3-year-old triploid specimens.

P–192 Efficacy of postponement of intracytoplasmic sperm injection timing after spindle visualization for Metaphase I oocytes

Abstract Study question Does postponement of intracytoplasmic sperm injection (ICSI) timing after spindle visualization for Metaphase I (MI) oocytes improve developmental outcomes of embryos? Summary answer Postponement of ICSI timing after spindle visualization for MI oocytes improves blastocyst utility rates. What is known already Immature oocytes are generally considered poor developmental outcomes. Meanwhile, the timing of ICSI adjusted by using spindle visualization can improve clinically utilized embryos and live birth rates, but these outcomes remain inferior to those of mature oocytes. In in vitro maturation culture, nuclear maturation is thought to occur before the completion of cytoplasmic maturation, and in immature oocytes, synchronization of nuclear and cytoplasmic maturation may be insufficient for ICSI immediately after spindle visualization. Study design, size, duration Data for this retrospective cohort study were obtained 672 oocytes retrieved under mild stimulation cycles using letrozole, in patients aged younger than 39 years between April 2017 and October 2020.Written informed consent was obtained from all patients. This study was approved by the institutional review board. Participants/materials, setting, methods As a control group, 464 MetaphaseIIoocytes that underwent ICSI immediately after visualization of the spindle were used. In group A, 103 MI oocytes underwent ICSI immediately after the first polar body release and spindle visualization, and in group B, 105 oocytes underwent ICSI 2–3 hours after spindle visualization. The primary outcomes were fertilization rates, degeneration, cleavage, embryo blastocyst formation, and utility rates. Outcomes were compared among the three groups. Main results and the role of chance The baseline fertilization rates of each group (control, A, B) were 82.3% (382/464), 73.8% (76/103), and 83.8% (88/105), respectively. The rate was significantly lower in group A than in the control group (P < 0.05), and also tended to be lower in group A than in group B, although the difference was not significant. There was no significant difference in abnormal fertilization rates, oocyte degeneration rates, cleavage rates, and blastocyst formation rates among the three groups. [control, A, B: abnormal fertilization rate: 4.3% (20/464), 8.7% (9/103), 4.8% (5/105); oocyte degeneration rates: 3.0% (14/464), 1.9% (2/103), 3.8% (4/105); cleavage rates: 95.6% (307/321), 93.8% (61/65), 98.7% (74/75); blastocyst formation rates: 58.6% (177/302), 51.7% (31/60), 55.4% (41/74), respectively]. The blastocyst utility rates of control group and group B were significantly higher than in group A [41.7% (126/302), 45.9% (34/74), 26.7% (16/60), respectively] (P < 0.05). There were no significantly different outcomes between the control group and group B. Limitations, reasons for caution The optimal timing of ICSI for MI oocyte cannot be determined by the presence or absence of spindles. In addition, the postponement duration we set was based on reports which reported on final oocyte maturation, and further investigation is needed to establish the optimal ICSI timing for MI oocytes. Wider implications of the findings: In MI oocytes, postponement of ICSI timing after spindle visualization is essential for synchronization of the nucleus and cytoplasmic maturation. Trial registration number none

O-231 In vitro maturation of human immature (GV) oocytes after controlled ovarian hormonal stimulation with recombinant AMH in the maturation medium

Abstract Study question Does the addition of recombinant AMH to the in vitro maturation (IVM) medium improve the maturation of GV oocytes after controlled ovarian hormonal stimulation? Summary answer Our results show that the addition of recombinant AMH to the in vitro maturation medium improves the maturation rate of GV oocytes. What is known already Anti-Müllerian hormone (AMH) is an important hormone involved in the process of sex differentiation during embryonic development. At the transition to the 21. century, more and more researchers have studied the role of AMH in ovarian function, especially its impact on folliculogenesis. AMH is becoming one of the main biomarkers of ovarian reserve and ovarian-specific disease, however, little is known about its effect on human oocyte maturation. Therefore, we matured immature GV (germinal vesicle) oocytes in IVM medium with recombinant AMH to assess its effect compared to the conventional IVM procedure with FSH and hCG. Study design, size, duration In this two-year prospective study, we compared the maturation rate of four groups of immature (GV) oocytes matured in maturation medium with added i) AMH (n = 15), ii) AMH+FSH+hCG (n = 44), iii) FSH+hCG (conventional; n = 22), and iv) hormone-free maturation medium (control; n = 15). Each oocyte was matured in vitro for a maximum of 28 hours and monitored by time-lapse microscopy to assess the time of GV breakdown (MI) and extrusion of the polar body (MII). Participants/materials, setting, methods Ninety-six GV oocytes of 46 patients (aged < 38 years, involved in the ICSI programme) after short antagonist protocol of controlled ovarian hormonal stimulation were included after written informed consent. IVM of oocytes was performed in the MediCult IVM System (LAG and IVM medium, Cooper Surgical, Denmark) with added hormones, and in a CO2 incubator equipped with the PrimoVision time-lapse microscope (Vitrolife, Sweden). Main results and the role of chance IVM medium with added recombinant AMH gave the best result with all (100 %) oocytes matured in vitro. In conventional IVM medium with FSH and hCG, the oocyte maturation rate was poorer, with 68 % of oocytes matured in vitro. An even lower oocyte maturation rate (34 %) was observed in IVM medium with AMH, FSH and HCG, which might be explained by the antagonistic action of these hormones. In a group of control oocytes, 25 % of oocytes matured in vitro. The mean time to GV breakdown (MI stage) was 3.7 hours and to polar body release (MII stage) 20,5 hours. The time to MI stage was quite comparable in all groups of oocytes (3.5, 3.8 and 3.7 hours). There was a tendency for the polar body to be released later if AMH was added to the maturation medium (21.5 and 20.2 vs. 19.9 hours) but differences were not statistically significant, as revealed by Student’s t-test. In the control group of oocytes, these times were prolonged (4.2 and 22.2 hours) due to slow spontaneous maturation. These preliminary results demonstrate that AMH could directly affect the oocyte maturation in vitro. Limitations, reasons for caution The limitation is the relatively small number of oocytes included; GV oocytes accounted for less than 10 % of all oocytes in the in vitro fertilisation (ICSI) programme. Moreover, the proportion of GV oocytes spontaneously matured to MI stage before the start of the experiment and were therefore not included. Wider implications of the findings Based on our data, we believe that AMH directly affects human oocyte maturation in vitro. Despite the common knowledge that AMH regulates the recruitment of growing ovarian follicles, it appears that the addition of AMH to the maturation medium can improve the human oocyte maturation in vitro. Trial registration number 0120-546/2018/6

Updating the markers for oocyte quality evaluation: intracellular temperature as a new index.

BackgroundThe developmental competence of an embryo is principally dictated by the oocyte. Usually, oocyte selection is based on morphological properties; however, all morphological criteria that are currently used for the grading and screening of oocytes are not able to eliminate the subjectivity. Despite recent studies of the molecular factors related to oocyte quality, it is technically difficult to develop an index based on these factors, and new indices that reflect intracellular conditions are necessary.MethodsMorphological and molecular factors influencing developmental competence were comprehensively reviewed, and intracellular temperature was evaluated as a new marker of oocyte quality.Main findingsThe intracellular temperature of mature oocytes was high in fresh oocytes and decreased with time after polar body release. Under the same conditions, the intracellular temperature and its distribution differed among oocytes, suggesting that temperature represents the state of each oocyte.ConclusionIntracellular temperature is advantageous as an objective and quantitative indicator of oocyte quality. Further studies should evaluate the link between temperature and cellular phenomena to establish its use as an indicator of quality.

Effects of salinity and temperature on the timing of germinal vesicle breakdown and polar body release in diploid and triploid Hong Kong oysters, Crassostrea hongkongensis , in relation to tetraploid induction

Tetraploids are crucial important for commercial production of all-triploid Crassostrea hongkongensis. Successful production of tetraploid C. hongkongensis requires precise control of fertilization and applying treatment. The germinal vesicle breakdown (GVBD) ratio of stripped eggs before fertilization influences the efficiency of tetraploid inductions. According to our research, in both diploids and triploids, the time to 50% polar body 1 (PB1) release was significantly reduced if a GVBD ratio of 100% was reached before fertilization (p < 0.05). We consider the time at which there is 100% GVBD of stripped oocytes to be the optimum time point to start fertilization. 50% PB1 release is an important time point to apply the treatment for inducing tetraploids. Since lowering salinity (from 25 to 15 ppt) and increasing temperature (from 25 to 31°C) both accelerate the GVBD and the PB1 release rate of eggs, 31°C and 15 ppt are the most appropriate temperature and salinity level in which to perform tetraploid induction of C. hongkongensis. In these conditions, we found that there was significantly more variation in timing of 100% GVBD and 50% PB1 release among eggs from different triploids compared to different diploids (p < 0.05), creating difficulties in controlling fertilization and duration of treatment in tetraploid induction using triploid eggs. For reducing the variation, we recommend that treatments for tetraploid induction of C. hongkongensis should use eggs from individual triploid females.

Triploid or hybrid tetra: Which is the ideal sterile host for surrogate technology?

This work was aimed at developing an effective procedure to obtain sterile ideal host fish in mass scale with no endogenous germ cells in the germinal epithelium, owning permanent stem-cell niches able to be colonized by transplanted germ cells in surrogate technology experiments. Thus, triploids, diploid hybrids, and triploid hybrids were produced. To obtain hybrid offspring, oocytes from a single Astyanax altiparanae female were inseminated by sperm from five males (A. altiparanae, A. fasciatus, A. schubarti, Hyphessobrycon anisitsi, and Oligosarcus pintoi). Triploidization was conducted by inhibition of the second polar body release using heat shock treatment at 40 °C for 2 min. At 9-months of age, the offspring from each crossing was histologically evaluated to access the gonadal status of the fish. Variable morphological characteristics of the gonads were found in the different hybrids offspring: normal gametogenesis, gametogenesis without production of gametes, sterile specimens holding germ cells, and sterile specimens without germ cells, which were considered “ideal hosts”. However, only in the hybrid derived from crossing between A. altiparanae and A. fasciatus, 100% of the individuals were completely sterile. Among them 83.3% of the male did not present germ cells inside germinal epithelium, having only somatic cells in the gonad. The other 16.7% also presented spermatogonia inside the niches. Such a methodology allows the production of sterile host in mass scale, opening new insights for application of surrogate technologies.

Fertility and ploidy of gametes of allodiploid and allotriploid loaches produced by diploid Misgurnus anguillicaudatus females and Paramisgurnus dabryanus males.

Artificial and natural hybridization of dojo loach Misgurnus anguillicaudatus (2N=50, DD for short) and large-scale loach Paramisgurnus dabryanus (2N=48, PP for short) are well-grown. However, these hybrid loaches have not yet been examined for fertility and ploidy of gametes. Here, histological observations, artificial propagation, observations of embryonic development, larval morphology, and ploidy analyses were conducted to determine the fertility and ploidy of gametes of allodiploid (DP for short) and allotriploid (DDP for short) loaches, produced by DD females × PP males and induced from fertilized eggs of DD females × PP males by cold shock to prevent the second polar body release, respectively. The ovaries of DP and DDP included smaller number of eggs when compared with those of the control DD, while full-grown oocytes were observed. Testes of these two loaches were delayed-developed without spermatids or mature spermatozoa. Results obtained here showed that DP and DDP were fertility-weakened female and sterile male. Moreover, DP females and DDP females could, respectively, produce few viable haploid eggs and few viable haploid and diploid eggs. This study will provide valuable information for fish hybrid researches.

The frequency of spontaneous triploidy in farmed Atlantic salmon produced in Norway during the period 2007-2014.

BackgroundSpontaneous triploidy has been reported in a number of fish species, and is often linked with in vivo or in vitro ageing of eggs post ovulation. Here, we provide the first investigation into the frequency of spontaneous triploidy in farmed Atlantic salmon by analysing more than 4000 fish from 55 farms, and approximately 1000 recaptured escapees, all sampled in the period 2007–2014. In addition, we compare microsatellite genotyping against flow cytometry and red blood cell diameter in a set of 45 putatively diploid and 45 putatively triploid Atlantic salmon.ResultsThe three methods implemented for ploidy determination gave consistent results, thus validating the methods used here. Overall, 2.0% spontaneous triploids were observed in salmon sampled on farms. The frequency of spontaneous triploids varied greatly among sea cages (0-28%), but they were observed in similar frequencies among the three primary breeding companies (1.8-2.4%). Spontaneous triploids were observed in all farming regions in Norway, and in all years sampled. Spontaneous triploids were also observed among the escapees recaptured in both the marine environment and in rivers.ConclusionsSpontaneous triploidy in commercially produced Atlantic salmon is likely to be a result of the practices employed by the industry. For logistical reasons, there is sometimes a pause of hours, and in some cases overnight, between killing the female broodfish, removal of her eggs, and fertilization. This gives the eggs time to age post ovulation, and increases the probability of duplication of the maternal chromosome set by inhibition of the second polar body release after normal meiosis II in the oocyte.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-015-0193-0) contains supplementary material, which is available to authorized users.

In vitro maturation of nude caprine oocytes recovered during oocyte collection

The present study was undertaken to investigate the effect of granulosa cell co-culture, granulosa monolayer and hormone on in vitro maturation of nude caprine oocytes. Nude oocytes (644) having evenly granulated cytoplasm were recovered by puncture technique. The oocytes were divided into 6 groups and matured in TCM-199 medium supplemented with 10% fetal bovine serum (FBS) and granulosa cell co-culture/ granulosa monolayer/ hormones. After maturation (27h), the oocytes were fixed for 15 min in 2.5% (w/v) glutaraldehyde in phosphate buffer saline,washed twice with PBS, stained with 0.1mg/ml DAPI in PBS and mounted on slides. The evaluation of nuclear status was done by epiflourescence microscope by observing either a polar body release or presence of two shiny chromatin spots. The maturation rate of oocytes in group 1, 2, 3, 4, 5 and 6 were 23.47, 32.40, 31.77, 26.00, 38.09 and 51.37%, respectively. The results of the study indicated that TCM-199 supplemented with hormones and coculture granulosa cells significantly increases the maturation rate of in vitro matured nude caprine oocytes.

Heterogeneous growth of the triploid Pacific oysters Crassostrea gigas created by chemical inhibition of polar body release

Triploids have several potential advantages over diploids in aquaculture, drawing an elevated commercial reaction into the realistic application of the techniques despite we are still in the early stage of triploid industry for the Pacific oysters Crassostrea gigas. We traced the growth performance of the triploid C. gigas for over a year from hatchery spats, which was created by manipulations of chemicals (Cytochalasin B, CB or 6-Dimethylaminopurine, 6-DMAP). The growth was clearly marked by an initial longer dormancy and following a great magnitude of heterogeneity. The dormancy was almost 9 to 10-month long or even longer and was considered as a downside of the creation. The heterogeneity was magnified by appearance of extraordinarily growing oysters in part during summer season, which could be a representative upside of the triploids. Overall, however, the results were not as positive as were expected. The longer dormancy and following heterogeneity observed in our practice could be marked as an additional negative sign of the chemical use. The present study, thus, might be highly indicative in the introduction of biological cross between tetraploid and diploid to produce natural triploid embryos.

Inhibiting sperm pyruvate dehydrogenase complex and its E3 subunit, dihydrolipoamide dehydrogenase affects fertilization in Syrian hamsters.

Background/AimsThe importance of sperm capacitation for mammalian fertilization has been confirmed in the present study via sperm metabolism. Involvement of the metabolic enzymes pyruvate dehydrogenase complex (PDHc) and its E3 subunit, dihydrolipoamide dehydrogenase (DLD) in hamster in vitro fertilization (IVF) via in vitro sperm capacitation is being proposed through regulation of sperm intracellular lactate, pH and calcium.Methodology and Principal FindingsCapacitated hamster spermatozoa were allowed to fertilize hamster oocytes in vitro which were then assessed for fertilization, microscopically. PDHc/DLD was inhibited by the use of the specific DLD-inhibitor, MICA (5-methoxyindole-2-carboxylic acid). Oocytes fertilized with MICA-treated (MT) [and thus PDHc/DLD-inhibited] spermatozoa showed defective fertilization where 2nd polar body release and pronuclei formation were not observed. Defective fertilization was attributable to capacitation failure owing to high lactate and low intracellular pH and calcium in MT-spermatozoa during capacitation. Moreover, this defect could be overcome by alkalinizing spermatozoa, before fertilization. Increasing intracellular calcium in spermatozoa pre-IVF and in defectively-fertilized oocytes, post-fertilization rescued the arrest seen, suggesting the role of intracellular calcium from either of the gametes in fertilization. Parallel experiments carried out with control spermatozoa capacitated in medium with low extracellular pH or high lactate substantiated the necessity of optimal sperm intracellular lactate levels, intracellular pH and calcium during sperm capacitation, for proper fertilization.ConclusionsThis study confirms the importance of pyruvate/lactate metabolism in capacitating spermatozoa for successful fertilization, besides revealing for the first time the importance of sperm PDHc/ DLD in fertilization, via the modulation of sperm intracellular lactate, pH and calcium during capacitation. In addition, the observations made in the IVF studies in hamsters suggest that capacitation failures could be a plausible cause of unsuccessful fertilization encountered during human assisted reproductive technologies, like IVF and ICSI. Our studies indicate a role of sperm capacitation in the post-penetration events during fertilization.

Post-ovulatory oocyte aging induces spontaneous occurrence of polyploids and mosaics in artificial fertilization of Japanese eel, Anguilla japonica

Spontaneous polyploids and mosaics have often been observed in artificially propagated larvae of the Japanese eel, Anguilla japonica. However, the mechanisms responsible for such unusual cytotypes are unclear. In this study, we examined the relationship of such polyploidization and mosaicisms in larvae resulting from artificial propagation to egg quality (fertilization rate and hatching rate) and viability of larvae, and then clarified the inducing factors and the mechanism for occurrence of such phenomena. Eggs stripped from females after induced maturation were artificially inseminated with sperm pre-cultured with artificial seminal plasma. Ploidy was determined by measuring the relative DNA content of the nuclei with flow cytometry. Of 968 embryos from 32 full-sib families, 9.1% were determined to be abnormal, most of which were triploids (86.5% of abnormal embryos); others were haploids (1.1%), aneuploids (2.3%), and mosaics (10.1%). The percentage of normal diploids from each family varied between 56.3% and 100% (90.9±11.7%, n=32). A significant positive correlation was found between the fertilization rate (P<0.001) or the hatching rate (P<0.001) and the percentage of diploids. Survival rate of triploid eels was similar to diploid eels at 10days after fertilization whereas aneuploids were inviable. When eggs were left in the body cavity of the female for four hours after ovulation and subsequently fertilized, the percentage of diploids decreased. We tried to elucidate the cause for the occurrence of spontaneous triploids by genetic analysis using 26 microsatellite DNA markers, which have been developed and mapped in relation to the centromere. These results suggest that the occurrence of cytogenetically unusual progeny is associated with over-ripening or aging of ova caused by the lapse of time from ovulation until fertilization, and spontaneous triploid larvae are derived from the duplication of the maternal chromosome set by inhibition of the second polar body release after normal meiosis I (crossing over) in oocyte and fertilization with normal sperm.

Motility, morphology, mitochondria and ATP content of diploid spermatozoa from sex-reversed clonal diploid and neo-tetraploid loach,Misgurnus anguillicaudatus

Summary Diploid spermatozoa were obtained from sex-reversed clonal diploid males and neo-tetraploid which derived from normal diploid × natural tetraploid cross followed by the 2nd polar body release inhibition. The spermatozoa from sex-reversed clonal diploid males exhibited a significant reduction in motility when compared with those from normal diploid and neo-tetraploid males. Electron microscopy revealed that diploid spermatozoa from sex-reversed clonal diploid and neo-tetraploid males exhibited normal shape similar to haploid spermatozoa from normal diploid males, except for larger sperm-head size and longer flagellum. Considering flow-cytometric results, volume of mitochondrial mass per spermatozoon was also increased in proportion to the elevation of ploidy status from haploid to diploid sperm. Diploid spermatozoa from neo-tetraploid increased the number of mitochondria and the ATP content, but those from sex-reversed clonal diploid males did not show such results.

Cytological studies on induced meiogynogenesis in Japanese flounderParalichthys olivaceus(Temminck et Schlegel)

The cytological process of induced gynogenetic development and subsequent chromosome duplication by a cold shock treatment was observed in Japanese flounder Paralichthys olivaceus (Temminck et Schlegel). Mature eggs were at the metaphase of the second meiosis when inseminated with ultraviolet (UV)-irradiated sperm of red sea bream Pagrus major. After the beginning of cold shock treatment, the previously visible spindle became invisible, probably due to the side effect caused by cold shock treatment. The chromosomes at the centre of the metaphase plate were condensed. This condition continued during the duration of the cold shock treatment and several minutes after it. The release of the second polar body was blocked and it developed into a female-like pronucleus. Then, it fused with the female pronucleus to generate a diploid zygotic nucleus, and the egg exhibited the first mitosis. Consequently, the haploid female chromosome set of the egg was doubled by the inhibition of the second polar body release. There was a significant delay in developmental time in the gynogenetic eggs when compared with that in the normal eggs. From the time of insemination to early cleavage, the UV-irradiated heterospecific sperm nucleus remained condensed.

Novel method for the nuclear transfer of adult somatic cells in medaka fish (Oryzias latipes): Use of diploidized eggs as recipients

Until recently, the nuclear transfer of adult somatic cell nuclei in fish has been unsuccessful. This is primarily because of chromosomal aberrations in nuclear transplants, which are thought to arise due to asynchrony between the cell cycles of the recipient egg and donor nucleus. We recently succeeded in circumventing this difficulty by using a new nuclear transfer method in medaka fish (Oryzias latipes). Instead of enucleated eggs, the method uses non-enucleated and diploidized eggs, obtained by retention of the second polar body release, as recipients in the nuclear transfer of primary culture cells from the caudal fin of an adult green fluorescent protein gene (GFP)-transgenic strain. We found that 2.7% of the reconstructed embryos grew into diploid and fertile adults exhibiting donor expression characteristics and transmission of the GFP marker gene to progeny. The mechanism underlying the generation of nuclear transplants using this method is unknown at present; however, analyses of donor and recipient nuclei behavior and the cytoskeletal mechanisms involved in the early developmental stages, as well as the special ability of diploidized eggs to facilitate reprogramming of the donor nuclei will result in elucidation of the mechanism.

Diploidized eggs reprogram adult somatic cell nuclei to pluripotency in nuclear transfer in medaka fish (Oryzias latipes)

Reprogramming of adult somatic cell nuclei to pluripotency has been unsuccessful in non-mammalian animals, primarily because of chromosomal aberrations in nuclear transplants, which are considered to be caused by asynchrony between the cell cycles of the recipient egg and donor nucleus. In order to normalize the chromosomal status, we used diploidized eggs by retention of second polar body release, instead of enucleated eggs, as recipients in nuclear transfer of primary culture cells from the caudal fin of adult green fluorescent protein gene (GFP) transgenic medaka fish (Oryzias latipes). We found that 2.7% of the reconstructed embryos grew into adults that expressed GFP in various tissues in the same pattern as in the donor fish. Moreover, these fish were diploid, fertile and capable of passing the marker gene to the next generation in Mendelian fashion. We hesitate to call these fish 'clones' because we used non-enucleated eggs as recipients; in effect, they may be chimeras consisting of cells derived from diploid recipient nuclei and donor nuclei. In either case, fish adult somatic cell nuclei were reprogrammed to pluripotency and differentiated into a variety of cell types including germ cells via the use of diploidized recipient eggs.

Induction of tetraploid gynogenesis in the European sea bass (Dicentrarchus labrax L.).

A preliminary study on tetraploid gynogenetic induction in the European sea bass was performed by pressure-blocking the second polar body release and the first cleavage in eggs fertilized with ultraviolet-irradiated sperm. Fertilization of eggs with genetically inactivated sperm produced only haploid development that terminated around hatching. Pressure treatments (8.500 psi for 2 min) applied at 6 and 65 min after fertilization (a.f.) produced variable levels (7-95%) of tetraploid larvae at hatching. A small proportion of mosaics (3.8n/4.2n) was also recorded.

Delayed meiosis and polar body release in eggs of triploid Pacific oysters, Crassostrea gigas, in relation to tetraploid production.

The dynamics of polar body release are important for creating polyploid shellfish. For producing triploids, these dynamics concern meiosis in diploid eggs and are well understood. For creating tetraploids, eggs from triploids are employed and the dynamics, variation, and environmental influences upon polar body release are less studied. We investigated the effects of several agents on the timing of 50% first polar body (PB1) release in eggs of triploids. PB1 release is generally slower in triploid eggs than diploid ones at 26 degrees C. Lowering the temperature (from 26 to 19 degrees C) had a marked effect on timing of 50% PB1 in both diploid and triploid eggs. While lower temperature merely slowed development in diploid eggs, it nearly halted it in triploid eggs. At any temperature, the variability in 50% PB1 release was much higher in triploid eggs than diploid ones; this variation occurred both within eggs from individual females and among eggs from different females. The amount of time eggs remain in seawater between the time they are stripped and fertilized (or time of hydration) also affected rate of meiosis. In triploid eggs, the average time necessary for the expulsion of 50% PB1 was 23 min post-fertilization (PF) for 75 min of hydration versus 29 min PF for 35 min. However, increasing the time of hydration had no effect on the variability in the timing among females. Serotonin also had no effect on the dynamics of polar body release in triploids. Variability among triploid females in timing of meiosis cannot be improved with any treatments we tried. Consequently we recommend that treatments of triploid eggs to produce tetraploids incorporate a single female at a time.

Clonal nature of gynogenetically induced progeny of triploid (diploid × tetraploid) loach,Misgurnus anguillicaudatus (pisces: cobitididae)

In the loach Misgurnus anguillicaudatus, triploid fish, which had been produced by hybridizations between normal diploid females and natural tetraploid males, laid mature hexaploid (triploid egg nucleus + triploid polar body nucleus) eggs of large size as well as diploid (haploid egg nucleus + haploid polar body nucleus) eggs of small size. Gynogenetic triploidy was artificially induced by activating the large eggs with UV-irradiated spermatozoa. Gynogenetic hexaploidy was also generated by blocking the polar body release with pressure shock after induction of gynogenesis. Unexpected mosaics and pentaploid gynogens were infrequently observed in these crosses. Most triploid and all hexaploid gynogens from each triploid female demonstrated absolutely identical DNA fingerprints, suggesting clonal nature among the mature large eggs of each female. However, a small number of triploid gynogens were aclonal with slightly different DNA fingerprints. These results indicate that triploid loach generate viable large eggs which are genetically identical or highly similar to the somatic cells. J. Exp. Zool. 278:412–421, 1997. © 1997 Wiley-Liss, Inc.