Embryo Damage Research Articles

Comparison of development and implantation of human embryos biopsied with two different methods: aspiration and displacement

To compare the development and implantation of human embryos biopsied with two different methods for preimplantation genetic diagnosis (PGD). Technique and method. A regional hospital IVF laboratory and private reproductive medicine clinic. Women undergoing IVF and PGD. Day 3 embryos were biopsied with aspiration and displacement; the embryos were cultured to blastocyst stage and then transferred. Blastocyst rate, pregnancy rate, and implantation rate. One hundred fifty-one embryos from 14 patients were biopsied with the blastomere displacement method and 51 embryos from 5 patients were biopsied with the aspiration method. Displacement used less time than aspiration; thus, the time of embryos exposed to biopsy solution was shorter when displacement was used. Blastocyst formation (55.6%-56.8%) and ongoing pregnancy rate (50%) were not different between the two biopsy methods. However, the implantation rate was significantly higher in patients with embryos biopsied using the displacement method (64.7%) than with the aspiration method (25%). Blastomere displacement uses less time and is an easy and simple method for embryo biopsy and could be used as an alternative method for embryo biopsy. Our results indicate that the displacement method minimizes embryo damage during biopsy that was indicated by a higher implantation rate.

Glutamine and hypotaurine improves intracellular oxidative status andin vitrodevelopment of porcine preimplantation embryos

We previously developed an in vitro-production system for porcine embryos and reported that the addition of glutamine (Gln) and hypotaurine (HT) during in vitro culture improved embryo development. This study examined the effects of Gln and HT on in vitro development, intracellular oxidative status and DNA damage of porcine preimplantation embryos. Porcine zygotes produced by in vitro maturation (IVM) and in vitro fertilization (IVF) were cultured until day 2 (day 0 = day of IVF) in porcine zygote medium (PZM) including 2 mM Gln and 5 mM HT, namely PZM-5. On day 2, the cleaved embryos were selected and cultured for 24 h in PZM-5 to which one of the following substances was added: (1) none (control); (2) Gln; (3) HT; or (4) Gln + HT. After 24 h of culture in each medium, the embryos were then returned to PZM-5 and cultured until day 5. Day-5 blastocyst yield was significantly higher in the Gln and Gln + HT groups (p < 0.05) than in the control and HT groups. In addition, Gln + HT significantly increased the total number of cells in blastocysts (p < 0.05) compared with the control. Although the number of cells and the intracellular GSH levels in day-3 cleaved embryos did not differ among treatments, addition of Gln, HT or Gln + HT significantly (p < 0.05) reduced the intracellular H2O2 content and the extent of DNA damage compared with the control. These results indicate that the presence of Gln and HT in PZM-5 from day 2 to day 3 promotes the development of porcine embryos by improvement of intracellular oxidative status.

Effect of pentoxifylline in reducing oxidative stress-induced embryotoxicity

To 1) evaluate the embryotoxic effects of hydrogen peroxide on mouse embryo development and 2) examine if pentoxifylline can reverse hydrogen peroxide induced embryotoxicity. Prospective in vitro study examining the effects of varying concentrations of hydrogen peroxide and pentoxifylline on the blastocyst development rate alone as well as in combination. A dose-dependent decrease in % BDR was seen with increasing concentrations of H2O2. High concentrations of hydrogen peroxide (> 60 microM) were embryotoxic. Pentoxifylline (500 microM) was able to reduce the embryotoxic effect of hydrogen peroxide. Percent blastocyst development rate increased from 44% in hydrogen peroxide alone to 85% in hydrogen peroxide and pentoxifylline coincubation. Pentoxifylline may be beneficial in reducing hydrogen peroxide induced embryo damage and improve IVF outcome. Patients with endometriosis-associated infertility may benefit from the use of pentoxifylline without significantly affecting embryo development.

Effects of β-mercaptoethanol and cycloheximide on survival and DNA damage of bovine embryos stored at 4 °C for 72 h

The objectives of this study were to determine the effects of cycloheximide (CHX) and β-mercaptoethanol (β-ME) during storage of in vitro-produced (IVP) bovine blastocysts for 72 h at 4 °C on their survival, hatching capacity and DNA damage. In Experiment 1, when blastocysts were stored in a medium supplemented with 25, 50 or 100 μg/mL of CHX, or 25, 50 or 100 μM of β-ME, the blastocysts stored with 25 μg/mL of CHX had a significantly higher survival rate than that of the blastocysts stored without CHX (79.5% versus 54.2%). In contrast, β-ME had no apparent effects on the survival and hatching capacity of stored embryos. In Experiment 2, to investigate synergistic effects of CHX and β-ME during storage of blastocysts on their developmental parameters and DNA damage, they were stored in the medium with CHX (25 μg/mL) and β-ME (50 μM). The combination of CHX and β-ME had no significant effects on the survival of blastocysts. The proportion (6.8%) of DNA-fragmented cells in the blastocysts stored with CHX was similar to that (5.4%) in the non-stored blastocysts (positive control) and significantly lower than that (9.7%) in the blastocysts stored without CHX and β-ME (negative control). However, there were no significant differences among the proportions of dead cells of blastocysts in the storage groups. Therefore, the supplementation of CHX in the storage medium had a beneficial effect on the proportions of survival and DNA-fragmented cells in the stored embryos, whereas the β-ME alone or in combination with CHX had no positive effects on either of these proportions.

145 STAGE-SPECIFIC EFFECT OF OXIDATIVE STRESS ON DEVELOPMENTAL COMPETENCE, ROS GENERATION AND DNA DAMAGE OF PORCINE PARTHENOGENETIC EMBRYOS

We investigated the effect of oxidative stress on stage specific developmental ability, reactive oxygen species (ROS) generation and DNA damage of parthenogenetically activated porcine embryos. Cumulus-oocyte complexes (COCs) were aspirated from follicles on the surface of ovaries. The COCs were matured in NCSU-23 containing 10% (vol/vol) porcine follicular fluid and 10 IU/mL hCG during the first 22 h followed by an extra 22 h of culture in the hormone free NCSU-23. After 44 h of maturation, oocytes were denuded of cumulus cells and used for activation. Oocytes were activated by a 100-�sec pulse of 1.5 kV/cm DC with 1-mm electrodes in 0.3 m mannitol, 0.1 mm MgSO4, and 0.05 mm CaCl2. Activated oocytes were then cultured for 5 h in NCSU-23 containing 5 mg/mL BSA, 10 �g/mL EGF and 7.5 �g/mL cytochalasin B. Embryos were then cultured for 6 days in PZM-5. In Experiment 1, after parthenogenetic activation, embryos were cultured at 38.5�C under 5% O2, 5% CO2 and 90% N2 (defined as 5% O2) or 5% CO2 in air (20% O2). The oxygen concentration for embryo culture was changed from 5% to 20% on day 1, 2, 3, 4, and 5 post-activation, respectively. Embryos were also cultured throughout 6 days in 5 and 20% O2. About 100 embryos were used in each experiment. The number of embryos cleaved and developed to blastocyst stage was observed on day 2 and 6, respectively. In Experiment 2, 10 to 20 embryos cultured in 5 and 20% O2 were collected on Days 2, 4, and 6 for the detection of ROS, intracellular glutathione (GSH) levels and DNA damage. Intracellular ROS and GSH levels, were measured with fluorescent dyes (22,72-dichlorodihydrofluorescein diacetate for ROS and Cell Tracker" Blue for GSH). DNA damage of individual embryos was detected with a comet assay. DNA damage was quantified by measuring the length of the streak of DNA comet tail between the edge of the zona pellucida and the end of the visible comet tail by image analysis software. The rate of migrated DNA area per total DNA was also quantified. In Exp. 1, the rate of blastocyst formation was significantly decreased (P &lt; 0.001) when embryos were cultured for 6 days under 20% O2 (17.8 � 4%) than 5% O2 (38.5 � 5%). The rates of blastocyst formation were significantly decreased (P &lt; 0.05) when O2 concentration was changed from 5 to 20% before Day 3. After Day 4, high O2 concentration did not affect the development. In Exp. 2, relative ROS levels were significantly higher (P &lt; 0.05) on Day 2 (1.5 � 0.03) and Day 4 (1.4 � 0.06) in embryos cultured under 20% O2 than in those cultured under 5% O2 (1.0). No difference was observed in GSH level. DNA damage was significantly increased (P &lt; 0.05) in Day 2 embryos cultured under 20% O2 (161 � 54 �m) than 5% O2 (65 � 8.8 �m). These results indicate that the oxidative stress to embryo development by high O2 concentration is stage specific, that embryos are more sensitive in early stages, and that the oxidative stress has correlation with the increase of intracellular ROS and DNA damage.

Cold-induced imbibition damage of lettuce embryos: a study using cryo-scanning electron microscopy

The impact of rehydration on a multicellular organism was studied in lettuce (Lactuca sativaL.) embryos, using cryo-scanning electron microscopy (cryo-SEM). Naked embryos were sensitive to imbibitional stress, whereas embryos with an intact, thick-walled endosperm were not. Imbibitional injury to naked embryos was mainly confined to the outer 2–3 cell layers of the axis. These cells failed to swell and appeared poorly hydrated. Deeper layers were not affected even after extended periods of cold rehydration. The proportion of damaged cells (6–7% of total) roughly corresponded with the additional K+that gradually leached from the embryos. Damaged embryos were able to survive the loss of their surface layers and form adventitious roots. The swelling of inner tissues caused the dead surface layers to rupture into patches. Plasma membranes in dried embryos showed normal bilayer structure with a homogeneous distribution of intra-membrane particles (IMPs), also after non-injurious rehydration. Imbibitionally damaged plasma membranes showed many irregularities, such as globular insertions, that probably resulted from malfusions in the ruptured membrane, but the IMPs were still randomly distributed.

Randomized controlled study of human zona pellucida dissection using the zona infrared laser optical system: evaluation of blastomere damage, embryo development, and subsequent hatching

Objective To assess the effect of laser hatching on human embryo damage and subsequent development using the Zona Infrared Laser Optical System (ZILOS). Design Randomized controlled study. Setting Tertiary care fertility clinic. Patient(s) One hundred fourteen donated and discarded frozen human embryos. Intervention(s) Embryos were thawed, cultured with cleavage and morphology evaluated periodically, and randomized into control, partial hatching, or complete hatching groups. The laser hatching procedure was performed by ZILOS. Zona thickness and embryo diameter were recorded. Complete hatching involved the production of a full-thickness defect in the zona and partial hatching, a defect in the outer half of the zona. No laser treatment was administered to the control group. Main outcome measure(s) Blastocyst development and completion of hatching process. Result(s) No significant difference was noted between the three study groups for their baseline characteristics. There was no significant difference in blastocyst development among the three groups. However, the complete hatching group showed a significant increase in hatching compared to the control group. Conclusion(s) Complete laser hatching of human embryos using the ZILOS does not have an adverse effect on subsequent development and increases the rate of completion of hatching.

Effect(s) of assisted hatching on assisted conception (IVF & ICSI).

Failure of implantation and thus conception might result from inability of the blastocyst to escape from its zona pellucida. Artificial disruption of this coat has been proposed as a method of improving the success of assisted conception. To determine whether assisted hatching of embryos facilitates live births, clinical pregnancy and implantation and whether it impacts on negative outcomes (such as miscarriage). We searched the Cochrane Menstrual Disorders and Subfertility Group trials register (26 November 2002), the Cochrane Controlled Trials Register (Cochrane Library Issue 2, 2002), MEDLINE (1996 to February 2003), EMBASE (1980 to February 2003) and reference lists of articles. Authors were contacted for missing and/or unpublished data. Trials were identified and independently screened by two reviewers. Randomised controlled trials of AH (mechanical, chemical or laser disruption of the zona pellucida prior to embryo replacement) versus no AH that reported live birth, clinical pregnancy or implantation rates were included. Qualitative assessments and data extraction were performed independently by two reviewers. Outcomes were extracted as rates and combined using random effects meta-analysis, sensitivity analysis, subgrouping and meta-regression where appropriate. Twenty-three randomised controlled trials (2572 women) were included. There was no significant difference in the odds of live births in the AH compared with control groups (6 RCTs; OR 1.21, 95% CI 0.82 to 1.78; 161 births from 523 women). Women undergoing assisted hatching were significantly more likely to achieve clinical pregnancy (722 clinical pregnancies in 2175 women, OR 1.63, 95% CI 1.27 to 2.09), however the studies were heterogeneous. Implantation data were difficult to analyse due to the practice of replacing multiple embryos in individual women. The trials provided insufficient data to investigate the impact of assisted hatching on several important outcomes, including monozygotic twinning, embryo damage, congenital and chromosomal abnormalities, and in vitro blastocyst development. There is insufficient evidence to determine any effect of AH on the 'take-home-baby rate' of assisted conception. There are also very few data regarding miscarriage rates and other adverse events. This prevents us from extrapolating the impact of AH on live births from our finding of improved odds of clinical pregnancy.

Antibodies to folate receptors impair embryogenesis and fetal development in the rat.

Folic acid (FA) supplementation reduces neural tube defects (NTDs) by 70%. However, the cause of most NTDs cannot be attributed to folate deficiency, to mutations of genes that encode folate pathway enzymes, and folate receptors (FRs) that mediate cellular folate uptake. Mouse embryos nullizygous for the ortholog of the FRalpha gene have lethal congenital abnormalities that are preventable by administration of folinic acid to the dams. To determine whether antibodies to FRs are similarly teratogenic, we studied a rat model. Immunohistochemistry with an antiserum to rat FRs was used to identify the receptors on reproductive tissues and embryos. Gestation day (GD) 8 rats received intraperitoneal injections of antiserum to the FRs, and their embryos were examined 2-9 days later. Some rats received pharmacologic doses of folinic acid or dexamethasone before the antiserum was administered. The FRs are present on oocytes, the oviduct, and uterine epithelial cells, and in the embryo at all stages examined between GD4 and GD15. The antiserum has a dose-related effect on embryo viability and organogenesis. Folinic acid prevented teratogenicity resulting from smaller doses of antiserum, but not that caused by larger doses. Resorption of embryos with the larger doses of the antiserum was prevented by dexamethasone. FRs are expressed on oocytes, epithelial cells of reproductive organs, and embryonic and extraembryonic tissues. Antiserum to FRs administered to pregnant rats causes embryonic damage. Embryo lethality with smaller doses of antiserum is preventable by administration of folinic acid, while larger doses cause embryo damage by immune-mediated cell lysis, which can be prevented by dexamethasone.

Polychlorinated -dibenzo-p-dioxins/furans and -dioxin-like biphenyls in eggs of common terns from lime island, St. Mary's river, Michigan, USA

Population collapse of common tern (Sterna hirundo) from Lime Island in the St. Mary's River, Michigan, USA were related to the 2,3,7,8-chlorine substituted dioxins, furans, dioxin-like PCBs, and other possible factors using unhatch egg target contaminant analysis. The most toxic congeners, 2,3,7,8-TCDD/DF was found in all samples at noticeable concentrations. Magnitude of sum 2,3,7,8-PCDD/DFs were within the range of 39–93 pg/g wet wt. Dioxin-like PCBs were within the range of 360–1230 ng/g wet wt. Great Toxic Equivalent Quantity (TEQ) contributions by 2,3,7,8-TCDF, 2,3,4,7,8-PeCDF, 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD, and dioxin-like PCBs such as CB-77, CB-126 and CB-105 are considered greatly due to their influence of poor embryo development and consequent damage of embryos of common terns in egg injection studies. However, other organochlorines, heavy metals, and/or synergistic effects also taken in to account. On the whole, TEQs in the tern eggs were greater than the Lowest-Observable-Adverse Effect Level (LOAEL) for bald eagle (Haliaeetus leucocephalus) embryos observed in British Columbia, Canada. Concentrations of dioxin-like PCBs, dioxins, furans in the St. Mary's River food chain are at levels for concern for nesting colonial waterbirds. Eventually, the possible movements of analyzed chemicals downstream from a man-made flood event are discussed.

Hydrogen peroxide seed scarification of New Mexico collections of ribes cereum

Ribes cereum is an early-colonizing shrub species being evaluated for disturbed-land revegetation in New Mexico. Along with embryo dormancy, seed coat dormancy is thought to occur in this species, but acid scarification has failed to improve germination consistently. To examine the efficacy ofscarification using hydrogen peroxide, seeds from six R. cereum collections from throughout New Mexico were soaked in 3% hydrogen peroxide for 0, 4, 8, or 16 hours and then stratified for 0,60,90 or 120 days. For all collections, a four-hour soak improved germination. The combination of a four-hour hydrogen peroxide soak followed by 120 days of stratification was the optimal treatment combination for most collections. With the exception of the southernmost collection, stratification was more effective than hydrogen peroxide scarification in promoting R. cereum germination. For this small-seeded species, a four-hour soak in 3% peroxide effectively degrades seed coat tissues without causing embryo damage. The choice between sulfuric acid and hydrogen peroxide as chemical scarification agent is discussed in terms seed size, seed coat thickness and seed susceptibility to damage.

Effect of oxidative stress on development and DNA damage in in-vitro cultured bovine embryos by comet assay

The correlation of oxidative stress on development and DNA damage in bovine embryos was investigated by the comet assay (single-cell microgel electrophoresis), an effective technique for detecting single-strand DNA breakage. After in vitro maturation and fertilization, one-cell stage embryos without cumulus cells were cultured for 8 days in SOF medium containing amino acids plus 5 % FCS under low (5%) and atmospheric (20 %) oxygen concentration. After 8 days of culture, the extent of blastocyst formation was significantly decreased (P<0.001) when embryos were cultured under 20 % oxygen concentration (5.8 ± 2.4 %) when compared to embryos cultured under 5 % oxygen concentration (35.1 ± 6.7 %). At the day 3 of development, DNA damage of individual embryos cultured under 5 % or 20 % oxygen concentration was measured by the comet assay, which entails microgel electrophoresis that can readily detect damaged DNA. After measuring the DNA damage in individual embryos by the comet assay, the length (149.9 ± 15.3 μm) of the migrating DNA fragment that is indicative of damaged DNA was significantly increased (P<0.001) in the embryos cultured under 20 % oxygen concentration when compared to embryos cultured in 5 % oxygen concentration (42.3 ± 7 μm). The length of damaged DNA in more than 50 % of embryos was less than 50 μm. when embryos were cultured under 5 % oxygen concentration. In contrast, the distribution of damaged DNA shifted to the more damaged extent when embryos were cultured under 20 % oxygen concentration. These results demonstrate that the retardation in bovine embryo development than in likely due oxidative stress as a consequence of the higher atmospheric oxygen concentration is positively correlated with an increase in the extent of DNA damage. Moreover, these results demonstrate that the comet assay is a useful method to evaluate embryo culture conditions.

A developmental timer that regulates apoptosis at the onset of gastrulation

Recent work identified an apoptotic program in gastrulation stage Xenopus embryos (Anderson, J.A., Lewellyn, A.L., Maller, J.L., 1997. Mol. Biol. Cell 8, 1195–1206; Stack, J.H., Newport, J.W., 1997. Development 124, 3185–3195). Here, we characterize in detail this maternal cell death program, which is set up at fertilization and abruptly activated at the onset of gastrulation, following DNA damage or treatment of embryos with inhibitors of transcription, translation, or replication, between the time of fertilization and the midblastula transition (MBT). This apoptotic pathway is activated under tightly regulated developmental control(s): if the same treatments are applied after the MBT the apoptotic response is abrogated. Embryos displayed many characteristic apoptotic features, including DNA fragmentation, caspase activation, and embryonic death was blocked in vivo by the ectopic expression of Bcl-2, or injection of the caspase-3 inhibitor z-DEVD-fmk. The precise timing and the execution of this maternal cell death program is set at fertilization and does not depend on the type of stress applied, on cell cycle progression, or on de novo protein synthesis. This maternal developmental program might palliate the lack of cell cycle checkpoints in the pre-MBT embryo.

Early embryo loss is associated with the prior expression of macrophage activation markers in the decidua.

In early embryo loss, the activation of maternal immune effector mechanisms play a critical role in determining the success or failure of a pregnancy. We have previously shown that increased nitric oxide production by decidual macrophages is involved in early embryo loss occurring at day 12 of gestation. In this study, using reverse transcription-PCR and Southern blotting, the expression of inducible nitric oxide synthases (iNOS) and TNF-alpha mRNA was determined to quantify macrophage activation in individual murine embryos in a model of spontaneous early embryo loss. At day 8 of gestation, 32 and 29% of embryos with no apparent pathology showed an increase in iNOS and TNF-alpha mRNA expression, respectively. This corresponds to the natural resorption rate seen in the mouse model. In addition, the percentage of embryos with increased iNOS and TNF-alpha mRNA expression was further augmented when pregnant mice were induced to abort at a higher rate. These results showed, for the first time, a correlation between increased iNOS and TNF-alpha expression and embryo resorption. The results provide evidence for the presence of activated macrophages at implantation sites before overt embryo damage occurs.

Fracture Damage of Embryos and Its Prevention during Vitrification and Warming

The frequency of fracture damage in mouse blastocysts was examined by repeated cycles of vitrification and warming. Mouse blastocysts suspended in a solution of ethylene glycol, Ficoll, and sucrose in a straw were plunged into liquid nitrogen either directly (rapidly) or after holding them in liquid nitrogen vapor for 3 min or more (moderately). Vitrified samples were warmed by plunging them into 25°C water either immediately (rapidly) or after holding in air for 5–30 s (moderately). Warmed straws were recooled and rewarmed up to 9 times, to exaggerate the effect of cooling and warming. When embryos were cooled and warmed rapidly once, the incidence of the zona damage was only 1.2%, and 91% of recovered embryos reexpanded in culture. However, with repeated rapid cooling and warming, the incidence of zona damage increased, reaching 75% after 10 vitrifications; survival also dropped. When embryos were subjected to 10 cycles of moderate cooling and moderate warming with 15 or 30 s of suspension in air, 100% of the embryos had intact zonae. On the other hand, with moderate cooling followed by rapid warming or with rapid cooling followed by moderate warming, 41 and 16% of embryos had damaged zona, respectively, after 10 vitrifications. Therefore, fracture damage occurs during both cooling and warming, but it can be prevented completely by employing somewhat slower rates of cooling and warming. Furthermore, a high survival rate (88%) after 10 cycles of moderate cooling and moderate warming with 15 s of suspension in air indicates that vitrification, melting, or temperature fluctuation per se do not affect embryonic survival.

Embryonic and parental preferences for incubation temperature in herring gulls: implications for parent-offspring conflict

According to Trivers (1974), parent-offspring (P-O) conflict arises because offspring are selected to solicit more care than parents are selected to provide. However, should benefits fail to increase with increasing care, the offspring optimum can be reduced to the point where predicted P-O conflict vanishes. We examined offspring demand and parental care in such a benefit-limited system in herring gulls (Larus argentatus). In this species, parents typically neglect their lasthatched (C-) egg during the final hours of hatching (pipped-egg stage), allowing mean temperature to drop by about 4°C, to near 33°C. Other studies indicate that no increased offspring benefit arises from increasing pipped egg incubation temperature above that level, but embryo damage occurs if temperature drops lower. In such a system, P-O conflict over preferred incubation temperature is predicted to be minimal or absent. We assessed phenotypic manifestations of conflict by determining incubation temperature preferences of parent and offspring independently. Temperature provided solely by parental initiative was 33.9°C (artificial eggs, corrected for embryonic heat production). Preferred incubation temperature of pipped embryos was measured by exposing them to moderate chilling (20°C) punctuated by 4-min periods of rewarming when they called. Temperature of vocally thermoregulating embryos stabilized around a mean of 32.9–33.4°C, about 0.5–1.0°C below parental preference. Acting independently, parents and embryos each maintained egg temperature at or near minimum developmentally safe levels. Results provided no evidence for phenotypic conflict, as predicted by a benefit-limited version of Trivers' P-O conflict model. Benefit limitation may also be relevant to P-O conflict in other contexts such as feeding of newly-hatched young.

Possible role and mechanism of inactivation of decidual NK cells in pregnancy

Decidual NK cells are present in murine decidua in early pregnancy, but they decline by midpregnancy. The participation of these cells in rapid rejection of abnormal fetuses and possible cytolitic activity of these cells to trophoblasts seems unlikely. But, their production of cytokines (like CSF-1, which promote differentiation and growth of trophoblasts cells) may be the primary role of decidual NK cells in time of establishment of fetal-maternal connection. Later, inactivation of decidual NK cells by prostaglandins prevents damage of embryos.

Seed Predation by the Bean Weevil Acanthoscelides Obtectus on Phaseolus Species: Consequences for Seed Size, Early Growth and Reproduction

We examined potential implications of seed predation by the common bean weevil, Acanthoscelides obtectus for seed size variation in cultivated beans (Phaseolus vulgaris L., P. coccineus L. and P. lunatus L.) grown in common gardens. Significant seed size variation existed among different plants, among pods of differing seed number within plants, among successive seed crops within plants, and among seeds within pods. Within-plant sources of seed size variation were equally or more important than among-plant sources. Initial seed size strongly influenced early growth under laboratory and field conditions, and the advantage in early growth due to large seed size carried over to enhance subsequent plant reproduction in the field. Predationrelated shifts in seed and seedling size distributions apparently occurred without active selection by the bruchid. Oviposition that was evenly distributed among pods, but clumped within one or a few seeds within pods, allowed a greater proportion of seeds to escape from many-seeded pods. This, coupled with negative size/number relationships within and among plants, resulted in shifts toward smaller mean seed size. Due to nearly ubiquitous embryo damage by bruchid larvae, seed size of host plants was not related to tolerance to predation. We conclude that indirect sizeselective seed predation can have significant effects on maternal plant fitness both by altering the number of progeny produced, and by altering the fitness of surviving progeny.

When Is a Teratology Study Not an Evaluation of Teratogenicity?

The most widely used developmental toxicity safety evaluation is the Segment II protocol executed in rats and rabbits. It has come to be called the “teratology” study, even though it is not designed to produce terata and is not the optimal means for producing such, even if this should be the goal. To comply with the misstated letter of the Toxic Substances Control Act yet honor its intent to protect the conceptus from toxic insult, toxicologists are faced with terminology, definitions, and interpretations that result in unnecessary contention between the regulator and the regulated. When the developmental toxicology and teratology of the Segment II protocol are viewed in the appropriate context, the experiments provide not only a means for preventing embryo damage but also a means by which substances can be ranked according to their developmental hazard potential.

Effects of temperature and drying rate during dehydration of celery seeds on germination, leakage and response to gibberellin and cytokinin

A high temperature treatment of 32°C which prevents dehydration injury in celery seeds imbibed for 3 days at 17°C and then dried at 20°C, reduced leakage during rehydration, compared with seeds not given the high temperature treatment. Treatments which would normally release celery seeds from dormancy, such as low temperature imbibition or gibberellin (GA4/7) and benzyladenine (BA) applications had little effect on the germination of seeds exhibiting desiccation injury. However, GA4/7 did induce splitting of the seed coat and swelling of the endosperm, and this effect was enhanced by BA. It is suggested that in celery seeds high temperature prevents irreversible embryo damage, including membrane damage, caused by drying.