Unirradiated Embryos Research Articles

Survival fraction and phenotype alterations of Xenopus laevis embryos at 3 Gy, 150 kV X-ray irradiation

To determine the radiosensitivity of Xenopus laevis embryos, aquatic organism model, for toxicity studies utilizing X-rays at acute high dose levels, by analysing its survival fraction and phenotype alterations under one-exposure integral dose.We used the standard Frog Embryo Teratogenesis Assay Xenopus test during the early stages of X. laevis development. The embryos were harvested until st. 46 when they were irradiated. The radiation effects were checked daily for a week and the survival, malformations and growth inhibition were assessed. Sibling tadpoles as control organisms were used. Statistical analysis was performed to assess the extent of any damage. Irradiation was performed with an X-ray tube operated at 150 kV. The tube containing the tadpoles was exposed to an air kerma of 3 Gy as measured in air with an in-beam ionization chamber.After one week, survival fraction of irradiated embryos was 58%, while for control embryos it was 81%. Hence, irradiation with 150 kV, 3 Gy X-rays produced a 23% decrease of survival in regard to unirradiated embryos. The 70% of the irradiated embryos showed an altered distribution of the skin pigmentation, in particular on the dorsal area and in the olfactory pits, where the pigment concentration increased by a factor 2. In conclusion exposure of X. laevis to 3 Gy, 150 kV X-rays induced a reduction of embryos survival and a significant modification of pigmentation. The authors think that X. laevis embryos, at st 46, is a suitable biological model for large scale investigations on the effects of ionizing radiation.

Some properties of the signals involved in unirradiated zebrafish embryos rescuing α-particle irradiated zebrafish embryos

Purpose: The in vivo radiation-induced bystander effect (RIBE) and radiation-induced rescue effect (RIRE) induced between embryos of the zebrafish (Danio rerio) by alpha-particle irradiation were studied through the number of apoptotic signals revealed at 24 h post fertilization (hpf) through vital dye acridine orange staining.Materials and methods: RIBE and RIRE were verified through the significant increase and decrease in apoptotic signals in the partnered bystander and irradiated embryos, respectively.Results: The medium transfer experiment where irradiated zebrafish embryos were rescued through immersion in the medium previously conditioned by a larger number of irradiated zebrafish embryos showed (a) the involvement of a released stress signal in the induction of RIRE, and (b) RIBE and RIRE signals had the same function. With the help of 500 μM of the specific nitric oxide (NO) scavenger cPTIO (2-(4carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide), NO was confirmed as an essential signaling molecule for inducing both the RIBE and RIRE. On the other hand, the treatment with 20 μM of the carbon monoxide (CO) releasing chemical CORM-3 (tricarbonylchloro(glycinato)ruthenium (II)) suppressed the manifestations of RIBE but did not suppress RIRE.Conclusions: In conclusion, unirradiated zebrafish embryos need NO but not NO-induced damages to rescue α-particle irradiated zebrafish embryos.

Bystander Effect between Zebrafish Embryos in Vivo Induced by High-Dose X-rays

We employed embryos of the zebrafish, Danio rerio, for our studies on the in vivo bystander effect between embryos irradiated with high-dose X-rays and naive unirradiated embryos. The effects on the naive whole embryos were studied through quantification of apoptotic signals at 25 h post fertilization (hpf) through the terminal dUTP transferase-mediated nick end-labeling (TUNEL) assay followed by counting the stained cells under a microscope. We report data showing that embryos at 5 hpf subjected to a 4-Gy X-ray irradiation could release a stress signal into the medium, which could induce a bystander effect in partnered naive embryos sharing the same medium. We further demonstrated that this bystander effect (induced through partnering) could be successfully suppressed through the addition of the nitric oxide (NO) scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) into the medium but not through the addition of the CO liberator tricarbonylchloro(glycinato)ruthenium(II) (CORM-3). This shows that NO was involved in the bystander response between zebrafish embryos induced through X-ray irradiation. We also report data showing that the bystander effect could be successfully induced in naive embryos by introducing them into the irradiated embryo conditioned medium (IECM) alone, i.e., without partnering with the irradiated embryos. The IECM was harvested from the medium that had conditioned the zebrafish embryos irradiated at 5 hpf with 4-Gy X-ray until the irradiated embryos developed into 29 hpf. NO released from the irradiated embryos was unlikely to be involved in the bystander effect induced through the IECM because of the short life of NO. We further revealed that this bystander effect (induced through IECM) was rapidly abolished through diluting the IECM by a factor of 2× or greater, which agreed with the proposal that the bystander effect was an on/off response with a threshold.

α-Particle Irradiated Zebrafish Embryos Rescued by Bystander Unirradiated Zebrafish Embryos

We report data demonstrating that zebrafish embryos irradiated by α-particles can release a stress signal into the water, which can be communicated to the unirradiated zebrafish embryos sharing the same water medium, and then these unirradiated zebrafish embryos can release a feedback stress signal back to the irradiated embryos. The effects of radiation on the whole embryos were studied through quantification of apoptotic signals at 24 h post fertilization through staining with the vital dye acridine orange, followed by counting the stained cells under a microscope. We refer to this phenomenon as the "rescue effect", where the unirradiated embryos successfully helped the irradiated embryos mitigate the radiation induced DNA damages. The results showed that the number of apoptotic signals in the irradiated embryos was smaller when they were partnered with bystander unirradiated embryos in the same medium. The results also showed significantly fewer apoptotic signals in the irradiated embryos when the population of bystander embryos increased from 10 to 30, while keeping the population of irradiated embryos at 10. These data suggest that the stress communicated between the unirradiated zebrafish embryos and the irradiated embryos sharing the same medium will help "rescue" the irradiated embryos, and that the strength of the rescue effect depends on the number of rescuing bystander unirradiated embryos.

Radioadaptive Response Induced by Alpha-Particle-Induced Stress Communicated in Vivo between Zebrafish Embryos

We report data demonstrating that zebrafish embryos irradiated by alpha particles can release a stress signal into the water, which can be communicated to the unirradiated zebrafish embryos sharing the same water medium and thereby inducing a radioadaptive response in these unirradiated zebrafish embryos. The effects of radiation on the whole embryos were studied through quantification of apoptotic signals at 24 h post fertilization through staining with the vital dye acridine orange, followed by counting the stained cells under a microscope. In these experiments, dechorionated embryos were irradiated and then partnered with two other groups of unirradiated embryos, namely the bystander group (no more further treatments) and adaptive group (subjected to a further challenging dose) of embryos. The adaptive group of embryos were then separately further irradiated with a challenging dose. The results show that the number of apoptotic signals for the adaptive group is smaller than that for the corresponding control group, while that for the bystander group is larger than that for the corresponding control group. These suggest that the stress communicated in vivo between the irradiated zebrafish embryos and those unirradiated embryos sharing the same medium will induce radioadaptive response in the unirradiated embryos.

Mycosporine-like amino acids prevent UVB-induced abnormalities during early development of the green sea urchin Strongylocentrotus droebachiensis

Experiments were performed to determine how ultraviolet radiation (UVR) in the environmentally relevant range affects development of the sea urchin Strongylocentrotus droebachiensis (Muller) and whether mycosporine-like amino acids (MAAs), present in the early life stages, reduce UV-induced damage. Eggs, embryos, and larvae contained five MAAs having absorption maxima ranging from 320 to 334 nm. Eggs contained principally shinorine and porphyra-334, which absorb maximally at 334 nm and half-maximally at 312 and 348 nm, spanning much of the environmental range of biologically effective UVR. Concentrations of MAAs remained constant in unirradiated embryos through the gastrula stage, but decreased significantly in two-armed pluteus larvae. Daily exposure to combined photosynthetically active radiation (PAR, 400–700 nm) and UVR did not affect the concentration of MAAs in these embryos up to the two-armed pluteus stage. Prism larvae of sea urchins and the sand dollar Echinarachnius parma (Lamarck) did not accumulate shinorine from the surrounding seawater. Daily exposure of embryos to UVA (320–400 nm) and UVB (295–320 nm) radiation in the presence of PAR induced delays and abnormalities during development, and removing UVB eliminated this effect. Abnormalities in embryos included thickening of the blastoderm wall, filling of the blastocoel by abnormal cells, exogastrulation, and formation of abnormal spicules. The percentage of embryos that developed normally was lower in batches of embryos exposed to PAR + UVA + UVB, except in embryos from urchins maintained on MAA-rich diets. In all cases, the percentage of PAR + UVA + UVB-exposed embryos that developed normally was positively related to the concentration of MAAs in eggs from which the embryos developed. Thus, the MAAs found in S. droebachiensis embryos protect them against UVB-induced abnormalities during their development to at least the four-armed pluteus larval stage.

P element excision in Drosophila is stimulated by gamma-irradiation in transient embryonic assays.

The influence of gamma-irradiation on P element excision and excision-site repair mechanisms was directly tested by embryonic somatic excision assays. Preblastoderm P[ry+, delta 2-3](99B) embryos, having a stable source of somatically active P transposase, were irradiated previous to injection with P excision indicator plasmids. Frequencies of precise or nearly precise P excision increased with gamma-ray doses ranging from 0.5 to 3.5 Gy. Higher doses resulted in frequencies close to that in unirradiated embryos, though considerable embryonic lethality was also evident at these doses. A direct positive interaction between gamma-irradiation and P element activity is concluded.

Association of Protein Phosphorylation and Cell Cycle Progression after X-irradiation of Two-cell Mouse Embryos

Studies of protein phosphorylation in the second cell cycle of mouse embryos were assessed by [32P]-incorporation and one- and two-dimensional electrophoresis. Unirradiated and X-irradiated embryos were compared. X-irradiation in the G2 phase led to a block in this cell cycle phase. The phosphorylation of proteins with molecular weights of 30 and 46 kDa specifically correlated with the G2 phase-mitosis transition in normally dividing and in irradiated embryos. The time lag in the phosphorylation of these proteins between the unirradiated and X-irradiated embryos was identical to the duration of the radiation-induced G2 block. X-irradiation immediately decreased the phosphorylation of a 29 kDa protein to below detectable levels. Re-phosphorylation of this protein preceded the following mitosis. It is suggested that the 29 kDa protein may be involved in the regulation of events that led to the radiation induced G2 block and the initiation of mitosis after release.

A Chimera Embryo Assay Reveals a Decrease in Embryonic Cellular Proliferation Induced by Sperm from X-Irradiated Male Mice

Male mice were divided into three experimental groups and a control group. Mice in the experimental groups received one of three doses of acute X irradiation (1.73, 0.29, and 0.05 Gy) and together with the control unirradiated mice were then mated weekly to unirradiated female mice for a 9-week experimental period. Embryos were recovered from the weekly matings at the four-cell stage and examined by the chimera assay for proliferative disadvantage. Aggregation chimeras were constructed of embryos from female mice mated to irradiated males (experimental embryos) and embryos from females mated to unexposed males (control embryos) and contained either one experimental embryo and one control embryo (heterologous chimera) or two control embryos (control chimera). The control embryo in heterologous chimeras and either embryo in control chimeras were prelabeled with the vital dye fluorescein isothiocyanate (FITC), and the chimeras were cultured for 40 h and viewed under phase-contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution from the FITC-labeled embryo. Experimental and control embryos that were cultured singly were also examined for embryo cell number at the end of the 40-h culture period. In control chimeras, the mean ratio of the unlabeled cells:total chimera cell number (henceforth referred to as "mean ratio") was 0.50 with little or no weekly variation over the 9-week experimental period. During Weeks 4-7, the mean ratios of heterologous chimeras differed significantly from the mean ratio of control chimeras with the greatest differences occurring during Week 7 (0.41 for chimeras of 0.05 Gy dose group, 0.40 for chimeras of the 0.29 Gy dose group, and 0.17 for chimeras of the 1.73 Gy dose group). However, cell numbers of singly cultured experimental embryos differed from those of singly cultured control embryos for just Week 7 for the 0.29 and 1.73 Gy dose groups, even though the mean ratios of heterologous chimeras had differed significantly from those of homologous chimeras for 3 weeks prior to and 1 week following Week 7. We conclude that sublethal changes sustained by sperm in vivo from only 0.05 Gy of X irradiation can be inherited by the embryo as a proliferative disadvantage that becomes expressed if challenged by direct cell contact with an unirradiated embryo in an aggregation chimera.

Lectinhistochemical study of cell surface alterations in mouse embryos exposed to low radiation doses.

The peroxidase-coupled Dolichos biflorus agglutinin (DBA) served as a marker for cell surface alterations in embryonic mouse tissues exposed to low-dose radiation during the early organogenesis (day 9 post conception). In unirradiated embryos, DBA bound selectively to various organ primordia, depending on their differentiation state. The auditory vesicles and the developing blood vessels were the only tissues staining strongly with the lectin. The vascular endothelia also showed the highest radiosensitivity, expressed by the maximal reaction already at 12.5 cGy. Marked surface changes as well were registered in the basal part of the Rathke's pocket and in the roof of the diencephalon. After exposure to 25 cGy, a transient amplification of the reaction as compared with 12.5 cGy occurred firstly in the Rathke's pocket, then in the infundibulum and in auditory vesicles. The most distinct effects were achieved with 50 cGy. Remarkable is the prompt rise of the DBA-affinity in a narrow area of the myelencephalon, and subsequently also in the roof of the diencephalon. Furthermore, the infundibulum and the Rathke's pocket, both anlagen of the pituitary gland, bound heavily DBA during the entire examination period of 24 h. The present study demonstrated the outstanding suitability of the Dolichos biflorus agglutinin for histochemical detection of subtle cellular alterations by small radiation doses.

Anterior determinants in embryos of Chironomus samoensis: characterization by rescue bioassay

Embryos of Chironomus samoensis are programmed, by anterior u.v. irradiation, to form the abnormal body pattern 'double abdomen'. Most double abdomen embryos show a mirror-image duplication of abdominal segments in the absence of cephalic or thoracic segments. Such embryos can be 'rescued', i.e. restored to normal development, by microinjection of cytoplasm or RNA from unirradiated donor embryos. Most of the rescued embryos look completely normal and many of them hatch spontaneously. The rescuing activity decreases from the anterior to the posterior pole in the donor cytoplasm and must be delivered near the anterior pole of the recipient for maximum efficiency. Rescuing activity is present in total RNA extracted from whole, unirradiated embryos. Upon fractionation, the activity is associated with poly(A)+ RNA, with LiCl precipitate depleted of RNA smaller than 250 nucleotides (nt) and with a sucrose gradient fraction depleted of RNA larger than 500 nt. Corresponding fractions of RNA from Xenopus oocytes have no rescuing activity. The activity of Chironomus RNA is sensitive to u.v. irradiation with low fluence affecting less than 2% of the pyrimidine bases. Rescuing activity is present in cytoplasm until the blastoderm stage but disappears earlier from poly(A)+ RNA. Rescuing activity is also present, and localized, in cytoplasm of embryos from two related dipterans, Smittia sp. and Drosophila melanogaster, although the extent of rescue observed in Chironomus decreases with the phylogenetic distance between donor and recipient. The results of these and previous experiments indicate that dipteran embryos contain localized RNP particles acting as anterior determinants. In Chironomus, the activity of these particles seems to depend on the integrity of polyadenylated RNA of about 250 to 500 nt length.

An Assay Using Embryo Aggregation Chimeras for the Detection of Nonlethal Changes in X-Irradiated Mouse Preimplantation Embryos

We have developed a short-term in vitro assay for the detection of sublethal effects produced by very low levels of ionizing radiation. The assay utilizes mouse embryo aggregation chimeras consisting of one irradiated embryo paired with an unirradiated embryo whose blastomeres have been labeled with fluorescein isothiocyanate (FITC). X irradiation (from 0.05 to 2 Gy) and chimera construction were performed with four-cell stage embryos, and the chimeras were cultured for 40 h to the morula stage. The morulae were partially dissociated with calcium-free culture medium and viewed under phase contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution of irradiated (unlabeled) and control (FITC labeled) embryos per chimera. In chimeras where neither embryo was irradiated, the ratio of the unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.50 (17.8 +/- 5.6 cells per unlabeled embryo and 17.4 +/- 5.5 cells per FITC-labeled partner embryo). However, in chimeras formed after the unlabeled embryos were irradiated with as little as 0.05 Gy, the ratio of unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.43 (P less than 0.01). The apparent decreases in cell proliferation were not observed in irradiated embryos that were merely cocultured with control embryos, regardless of whether the embryos were zona enclosed or zona free. We conclude that very low levels of radiation induce sublethal changes in cleaving embryos that are expressed as a proliferative disadvantage within two cell cycles when irradiated embryos are in direct cell-to-cell contact with unirradiated embryos.

Ultraviolet effects on presumptive primordial germ cells (pPGCs) in Xenopus laevis after the cleavage stage

The presumptive primordial germ cell (pPGC) number with development after the cleavage stage and the fate of pPGCs damaged by uv irradiation were studied in successive Epon sections (0.5 μm thick) with the light microscope in both uv-irradiated and unirradiated Xenopus embryos. taking survival rate and sterility into consideration. The pPGCs of the uv-irradiated embryos occupy nearly the same location in the embryos as those of the unirradiated embryos at stages 12, 17, 23, and 28 [see Ikenishi, K., and Kotani, M. (1975). Develop. Growth Different. 17, 101–110 ]. At stage 33 34 they are found in the central part of the endoderm cell mass in the uv-irradiated embryos, while they are situated in the lateral or dorsal part of the endoderm cell mass in the unirradiated. In the uv-irradiated embryos, a cavity which was never found in the unirradiated embryos was observed in the endoderm cell mass beneath the archenteron cavity and in the almost-median part of the posterior endoderm cell mass at stages 17 and 23, respectively, and some vacuoles in pPGCs as well as in somatic cells around those pPGCs were noticed at stages 17– 33 34 . The number of pPGCs of the unirradiated enbryos increases about three- or fourfold during stages 12–46, while the pPGCs of the uv-irradiated embryos slowly increase in number from stage 17 to stage 28, indicating that the division occurs in pPGCs, then decrease with development and finally disappear from the tadpole.

Events in the germ cell lineage after entry of the primordial germ cells into the genital ridges in normal and u.v.-irradiatedXenopus laevis

ABSTRACTApproximately 20–25 primordial germ cells leave the endoderm between stages 38–41 and localize in the dorsal root of the mesentery by stage 43/44. At this time all the cells contain large quantities of yolk which is gradually resorbed. The cells begin dividing between stages 48–52. The number and size of the germ cells were measured in tadpoles between stages 48–54 of development. The results indicate that in females the germ cells divide more often than in males. In both sexes the mitoses are grossly unequal, leading to the formation of a new generation of germ cells which are considerably smaller (one-tenth to one-fifth) than the size of the primordial germ cells at stage 48. The germ cells in male tadpoles at stage 54 are larger than in female tadpoles at the same stage. In tadpoles which developed from eggs irradiated in the vegetal hemisphere with u.v. light at the 2- to 4-cell-stage, primordial germ cells migrate into the genital ridges much later (stage 46–48) than in unirradiated embryos. They also differ morphologically from germ cells in control animals at this stage in that they are approximately one-tenth the size, lacking yolk in the cytoplasm and have a more highly lobed nucleus. Comparison of the results in unirradiated and irradiated animals suggests that the germ cell lineage is composed of a series of ordered, predictable events, and serious disruption of one of the events deranges later events.

Repair of ultraviolet irradiation damage to a cytoplasmic component required for neural induction in the amphibian egg.

Localized ultraviolet irradiation of the amphibian egg destroys a cytoplasmic component that is required for neural induction. Destruction of that component severely diminishes the inducing capacity of the dorsal lip at gastrulation, as determined by embryological assays. Repair of the ultraviolet lesion can be achieved by replacing the dorsal lip of the irradiated embyro with a lip from an unirradiated embryo.

Actions of Gibberellic and Abscisic Acids on Lettuce Seed Germination Without Actions on Nuclear DNA Synthesis

Promotion and inhibition of DNA synthesis have been suggested as important causal events in the growth-promoting action of GA2 and the growthretarding action of ABA, respectively (11,12,15). We here report typical effects of GA and ABA during lettuce seed germination after seed irradiation that prevents detectable DNA synthesis as determined by 2 criteria: Feulgen microspectrophotometry and 3H-thymidine incorporation into nuclear DNA. Air-dry seeds of lettuce, Lactuca sativa 'New York,' were given 1.3 Mrad 60Co gamma-rays and then sown in Petri dishes containing distilled water or solutions of GA (Gibrel, Merck) or ABA (Shell Development Company) as indicated. Whereas lower radiation doses are sufficient to prevent mitosis during germination (7), the 1.3 Mrad dose was necessary to inhibit nuclear DNA synthesis, as will be shown in this report. In all cases the pH was 5.7, and the dishes were exposed to continuous illumination with 900 jUw/cm2 white light i(Sylvania GroLux) at 20°. After 5 days, germination of these irradiated seeds (as the percentage of total number of seeds sown) was 0 % in water controls, 84 % in 5 X 10-4 M GA, and 1.5 % in 5 X 10-4 M GA + 100 mg/l ABA. Thus germination of these seeds was very sensitive to GA and ABA. By Feulgen microspectrophotometry the relative amount of DNA per nucleus was measured for individual cells from ,(a) the apical 0.5 mm of the radicle of unirradiated embryos before sowing, and (lb) the protruded 1.1 mm of 5-day-old root tips from the

Radiation-induced free radicals in embryo and endosperm of barley kernels

Embryo and endosperm parts of barley kernels were irradiated at 77°K with 60Co γ-radiation, and ESR spectra recorded at 77°K immediately after irradiation or after heating to 201° or 293°K. In dry embryos (2% H 2O) mainly a narrow signal is obtained, which is relatively insensitive to heating. In dry endosperms (2% H 2O) a broad doublet is recorded which is changed into a narrow signal after only 3 min heating to 293°K. Unirradiated embryo material gives a (narrow) ESR absorption line, the amplitude of which decreases with increasing water content up to 15 per cent. No signal is detected in the case of pure endosperm material. The data so far are in agreement with earlier observations, which show a relationship between narrow-type radicals and biological damage.

Germ cell chimerism: absence in parabiotic frogs.

Embryos of the leopard frog deprived of primordial germ cells by treatment with ultraviolet light were joined in parabiosis with normal, unirradiated embryos. The irradiated member of the pair was not colonized by germ cells from its normal partner. Unlike the primordial germ cells of birds and mammals, the germ cells of frog embryos are not carried by the circulating blood.

Growth of embryos on synthetic media after excision from gamma-irradiated (525 kr) and unirradiated wheat grains

Embryos exclusive of scutella were excised from γ-irradiated (525 kr) and unirradiated wheat grains and cultured in darkness on defined media. Growth of the embryos from irradiated grain occurred without detectable cell divisions. Absence of a nitrogen source reduced growth of irradiated and unirradiated embryos to similar extents. Absence of carbohydrate completely prevented detectable growth in both irradiated and unirradiated embryos. The growth responses of the irradiated embryos to single and mixed carbohydrates were similar to the growth responses of unirradiated embryos in the following respects: (1) dependency of growth on d-glucose concentration, (2) growth on either sucrose or fructose comparable with growth on d-glucose, (3) lesser but significant growth on either d-ribose or d-xylose, (4) incapacity to grow on d-galactose, d-mannose, or l-arabinose, and (5) reduced growth after addition of either l-arabinose or d-mannose to media containing a near optimal concentration of d-glucose. Similarly, addition of d-galactose to media containing a near optimal concentration of d-glucose reduced coleoptile and root growth of both irradiated and unirradiated embryos. By contrast, however, addition of d-galactose to media containing a near optimal concentration of d-glucose greatly reduced first leaf growth of the unirradiated embryos but had little or no effect on reducing the first leaf growth of the irradiated embryos. This differential effect of d-galactose on growth of the first leaf was the only major difference in gross growth characteristics found between the irradiated embryos growing without detectable cell division and the unirradiated embryos.

Occurrence of an inhibitor in wheat endosperm as revealed by embryo transplantation in irradiated seeds

Seeds (caryopses) of durum wheat var. Cappelli were soaked in water for 24 hr at 20°C to soften them enough for excision of embryos with scutellum. After irradiation of half of the soaked seeds (2–10 kr of X-rays), embryos were excised from both irradiated and unirradiated seeds and the following transplantations were made: (1) unirradiated embryo on irradiated endosperm, EM ( u) /EN ( i) and (2) unirradiated embryo on unirradiated endosperm, EM ( u) /EN ( u) (homotransplant, to serve as control). In 1962 and 1963, using seeds of the crops of 1961 and 1962 respectively, it was demonstrated that endosperms of after-ripened seeds stimulated growth in the EM ( u) /EN ( l) seedlings. Evidence was found that this stimulatory effect results from the destruction—or inactivation—by radiation of an inhibitor present in the endosperm of after-ripened seeds. The inhibitor is not present, in detectable amounts, in the endosperm of seeds during the total period of dormancy (first three months or more after harvest). The results are discussed in relation to the general problem of growth responses, including stimulation, in irradiated seeds.