An unusual radiation-induced G2 arrest in the zygote of the BALB/c mouse strain

Female mice of the BALB/c strain were superovulated, mated with males of the same strain, and irradiated with 1 Gy of X-rays at hourly intervals during the first cell cycle of the embryos. Two types of effects were found in the embryos, depending on the time of X-irradiation. When irradiation was delivered between 14 and 21 h after human chorionic gonadotrophin (hCG) injection, cultured two-cell embryos developed normally up to the morula stage, where a high mortality occurred. On the other hand, when irradiation was given between 17 and 24 h after hCG injection, a high proportion of the eggs was unable to cleave and remained blocked at the one-cell stage. Cytofluorometric analysis of the pronuclear DNA content of uncleaved zygotes showed that DNA synthesis was unaffected by X-irradiation, and that they were blocked in G2 phase of the first cell cycle. Similar studies on other strains, as well as reciprocal crosses between BALB/c and F1(female BALB/c X male C57 BLACK) mice showed that the 'one-cell block' is determined by the maternal genotype and results most probably from a direct action of X-rays on a radiosensitive cytoplasmic factor necessary for the first embryonic cell division, and appearing 17 h after hCG injection. A high proportion of blocked zygotes (30-40 per cent) recovered partially, cleaved with a delay of about 20 h, and died soon after, almost none of them being able to reach the blastocyst stage. At the time of maximum radiosensitivity, the LD50 for development up to the blastocyst stage was 0.95 Gy.     

The X-ray induced G2 arrest in mouse eggs: a maternal effect involving a lack of polypeptide phosphorylation

Summary In some strains of mice, eggs when X irradiated during the pronuclear stage, undergo a mitotic block in the G₂ phase of the first cell cycle and cleave when the second division takes place in controls. The importance of this effect varies considerably with the strain and depends exclusively on the maternal genotype. In previous work, two-dimensional electrophoresis showed that eggs blocked at the one-cell stage after irradiation, undergo the same modifications in polypeptide synthesis as two-cell controls of the same age, except at the time of normal first mitosis, where three polypeptide sets of 30, 35 and 45 kDa appear only in cleaving controls. In the present study, we have found phosphorylations in dividing controls, on polypeptides of 30, 35 and 45 kDa. These phosphorylations are not seen in blocked irradiated eggs.     

Post-translational modifications of various polypeptides in relation to the first mitosis in BALB/c mouse ova

After X-irradiation at pronuclear stage, mice eggs from certain strains are reversibly blocked in G2 phase of the first cell cycle. However, evolution of polypeptide synthesis takes place in a normal way, except for three polypeptides sets of 30, 35 and 45 kd molecular weight that appear in dividing controls and not in blocked, irradiated eggs. It is shown that those first mitosis-specific polypeptides result from post-translational modifications, involving phosphorylations.     

Mouse one-cell embryos undergoing a radiation-induced G2 arrest may re-enter S-phase in the absence of cytokinesis

PCC (premature chromosome condensation) can be used for visualizing and scoring damage induced by radiation in the chromatin of cells undergoing a G1 or G2 arrest. A method involving the fusion of irradiated single embryonic cells with single MI oocytes was used to induce PCC in mouse zygotes of the BALB/c strain, which suffer a drastic G2 arrest after X-irradiation (dose used 2.5 Gy). Other G2-arrested embryos were exposed in vitro to the phosphatase inhibitor calyculin A. Both methods furnished excellent chromosome preparations of the G2-arrested embryos. The mean number of chromosome fragments did not change significantly during G2 arrest, suggesting that zygotes of this strain are unable to repair DNA damage leading to such aberrations. Forty to fifty percent of the irradiated embryos were unable to cleave after G2 arrest and remained blocked at the one-cell stage for a few days before dying. PCC preparations obtained from such embryos suggested that about 30% of them had undergone a late mitosis not followed by cytokinesis and had entered a new DNA synthesis. These results are discussed in the light of recent observations in irradiated human cells deficient in the p53/14-3-3sigma pathway.     

Wound healing in X-irradiated mammalian skin.

Certain parameters of wound healing were investigated in mouse skin given 950 rads and 3325 rads of X-irradiation at various times relative to wounding. Increased inflammation, delayed dermal regeneration, and delayed contraction were noted in all irradiated groups. The activation of surrounding hair follicles, a process that usually accompanies wounding in mouse skin, occurred earlier, over a shorter elapsed time, and over a greater area of skin in animals irradiated prior to wounding than in the controls or in those irradiated and wounded simultaneously. Epidermal mitotic activity in wounds made at the time of irradiation was initially depressed but recovered by the second postoperative day. Wounds in pre-irradiated animals gave an unexpected result. They responded with an immediate burst of mitotic activity without the usual 24-hr lag that was seen in controls. In the pre-irradiated specimens a substantial number of cells appeared to die after dividing.     

Effects of X-irradiation on cell-cycle progression, induction of chromosomal aberrations and cell killing in ataxia telangiectasia (AT) fibroblasts

Survival, cumulative labeling indices, chromosomal aberrations and cell-cycle distribution by flow microfluorometry (FMF) were studied in fibroblasts from normal and three ataxia telangiectasia (AT) families after X-irradiation during density-inhibition of growth and immediate release by subculture to low density. Homozygotic AT (proband) fibroblasts were very hypersensitive to cell killing by X-irradiation (D0 = 40-45 rad). Fibroblasts from AT heterozygotes (parents) were minimally hypersensitive, with D0's (100-110 rad) slightly lower than those for normal fibroblasts (D0 = 120-140 rad). There were three different response groups for a G1 phase block induced by 400 rad of X-rays: (1) minimal or no G1 block was observed in AT homozygote cell strains; (2) 10-20% of the cells were blocked in G1 in normal cell strains; and (3) 50% or more of the cells were blocked in AT heterozygote strains. FMF profiles and cumulative labeling indices showed that homozygotic AT cells irradiated in plateau phase moved into the S-phase following subculture with no additional delay over non-irradiated controls. Homozygotic AT cells showed not only a 4-5 times higher frequency of X-ray-induced chromosomal aberrations than normal strains, but approximately 30% of these were of the chromatid-type. There were no differences in the frequency or type of X-ray-induced chromosomal aberrations between normal and heterozygotic AT cells.     

Anti-progesterone monoclonal antibody affects early cleavage and implantation in the mouse by mechanisms that are influenced by genotype

Pregnancy was blocked by anti-progesterone monoclonal antibody in two inbred (BALB/cJ, CBA/Ca) but to a lesser degree in an F1 hybrid (CBA/Ca male X BALB/cJ female) or an outbred (Tuck's no. 1) stock of mice when antibody was injected intraperitoneally (i.p.) at 32 h post coitum (p.c.) using a dosage of 9.5-10.9 nmol. This different antifertility effect could not be explained solely by altered tubal transport in inbred mice since the rate of transport was slightly accelerated in one stock (BALB/c) but not in another (CBA). In crossbred mice tubal transport was not significantly altered by antibody treatment. At Day 3 (54-58 h p.c.), the majority of embryos in control mice were at the 4-cell and 8-cell to morula stages in inbred and crossbred stock, respectively, but after antibody treatment they were mainly at the 4-cell stage in all 4 stocks. At Day 4 (78-82 h p.c.) the majority of embryos in control females had reached the blastocyst stage in all stocks, whereas after antibody treatment they had reached this stage in crossbred stock and relatively few had progressed so far in inbred stock. The results indicate that there are two events in early gestation which are susceptible to passive immunization with anti-progesterone monoclonal antibody. The first of these occurs during cleavage shortly after the 4-cell stage when embryo development was arrested in two inbred stocks of mice. Antibody effects on cleavage were not direct since embryos cultured in the presence of high concentrations of antibody, or antibody saturated with progesterone, continued to develop in the normal way and formed blastocysts. The second event is the onset of implantation, an effect also influenced by genotype. The decidual cell reaction induced by intraluminal oil injection was blocked by antibody injected at 8 or 32 h p.c. in BALB/c females, but only when injected at 8 h, and not at 32 h p.c., in F1 hybrid females. The results show that there is a greater resistance in two crossbred stocks compared with two inbred stocks to the effects of passive immunization against progesterone in early pregnancy.     

Effect of Initiation Time of Hydrostatic Pressure Shock on Chromosome Set Doubling of Tetraploidization in Turbot <Emphasis Type="Italic">Scophthalmus maximus</Emphasis>

The objective of the study was to clarify the effects of initiation time on chromosome set doubling induced by hydrostatic pressure shock through nuclear phase fluorescent microscopy in turbot Scophthalmus maximus. The ratio of developmentally delayed embryo and chromosome counting was used to assess induction efficiency. For the embryos subjected to a pressure of 67.5 MPa for 6 min at prometaphase (A group), chromosomes recovered to the pre-treatment condition after 11-min recovering. The first nuclear division and cytokinesis proceeded normally. During the second cell cycle, chromosomes did not enter into metaphase after prometaphase, but spread around for about 13 min, then assembled together and formed a large nucleus without anaphase separation; the second nuclear division and cytokinesis was inhibited. The ratio of developmentally delayed embryo showed that the second mitosis of 78% A group embryo was inhibited. The result of chromosome counting showed that the tetraploidization rate of A group was 72%. For the embryos subjected to a pressure of 67.5 MPa for 6 min at anaphase (B group), chromosomes recovered to the pre-treatment condition after about 31-min recovering. Afterwards, one telophase nucleus formed without anaphase separation; the first nuclear division was inhibited. The time of the first cleavage furrow occurrence of B group embryos delayed 27 min compared with that of A group embryos. With the first cytokinesis proceeding normally, 81.3% B group embryos were at two-cell stage around the middle of the second cell cycle after treatment. Those embryos were one of the two blastomeres containing DNA and the other without DNA. The first nuclear division of those embryos was inhibited. During the third cell cycle after treatment, 65.2% of those abovementioned embryos were at four-cell stage, cytokinesis occurred in both blastomeres, and nuclear division only occurred in the blastomere containing DNA. Of those abovementioned embryos, 14.0% were at three-cell stage and cytokinesis only occurred in the blastomere containing DNA. The result of chromosome counting showed that the tetraploidization rate of B group was only 7%. To summarize what had been mentioned above, mechanisms on chromosome set doubling of tetraploid induction would be different with different initiation time of hydrostatic pressure treatment. Chromosome set doubling was mainly due to inhibition of the second mitosis when hydrostatic pressure treatment was performed at prometaphase. Otherwise, chromosome set doubling was mainly due to inhibition of the first nuclear division when hydrostatic pressure treatment was performed at anaphase. Induction efficiency of tetraploidization resulted from inhibition of the second cleavage was higher than which resulted from inhibition of the first nuclear division. This study was the first to reveal biological mechanisms on the two viewpoints of chromosome set doubling through effect of initiation time of hydrostatic pressure treatment on chromosome set doubling in tetraploid induction.     

Cell cycle dependence of transformation expression in mouse cells transformed by a thermosensitive mutant (Ts-121) of polyoma virus.

Change in division capability as a phenotypic expression of cellular transformation was investigated by using one of the temperature-sensitive (ts) mutants of the polyoma virus-transformed cell line, the 121-6-5 cells of BALB/3T3. When contact -inhibited cells were treated with hyaluronidase at 39 degrees C, a single round of cell division was induced after which cell growth was inhibited by cell density. However, if the cells were incubated at 35 degrees C, after the enzyme treatment, density-inhibition block disappeared and the cells entered a second division. This indicates that the release of cells from density-inhibition depends on the low temperature incubation. The ability of cells to complete a second division was examined by shifting the cells from 39 degrees C to 35 degrees C during different phases of the first division cycle after the enzyme-treatment. A 6-hour incubation of S phase cells at 35 degrees C resulted in a second cycle of division, while the 24-hour incubation of G1 cells at 35 degrees C did not induce a second round of division. These results suggest that expression of the transformed phenotype in 121-6-5 cells is clearly dependent upon both the temperature and the phase of the division cycle.     

Neutron irradiation of late mouse embryos (15-19 days) in utero

Pregnant female C57B1/6 mice were irradiated with a single whole-body dose of 0.5 Gy neutrons. The F1 hybrid embryos were exposed to the neutrons in utero on Day 17 +/- 2 of gestation. 178/439 (40.6%) of the irradiated fetuses and 26/217 (12%) of the control mice died within 2 weeks after birth. In both irradiated and control mice, most deaths (95 and 77%, respectively) occurred within 3 days of birth: most animals in both groups died on Day 2. There was no significant difference in the number of living young born per litter (7.2) between the neutron-irradiated mothers and their unirradiated controls. The irradiated mice weighed significantly less than their controls. On the first day after birth, body weights of mice irradiated in utero averaged only 85% of control weights. Body weights did not reach control levels until 6 months after birth. Several organs were weighed at regular intervals in both irradiated and control mice. Spleens and thymus glands showed no significant differences between the two groups. The livers and kidneys of the irradiated mice weighed slightly less than their controls. The brain weight of 21-day-old neutron-irradiated mice was 30-35% less than control brains. The weight loss of the brain was not only a relative loss, but also an absolute one, based on brain weight/body weight ratios. Histological analysis of the central nervous system showed pycnotic nuclei, inhibition of mitosis in neuroblasts, and cell death in the irradiated brains. The weight reduction of the brain was not due to water loss. Our hypothesis is that the early mortality after birth is related to the killing of the radiation-sensitive neuroblasts. When newborn mice (1-7 days old) were irradiated in vivo with the same neutron dose of 0.5 Gy, neither the reduction in brain weight nor the early mortality was observed. The early deaths of the neutron-irradiated mouse embryos does not appear to be caused by either the hematological or the gastrointestinal radiation syndrome.     

Muscle development in vitro following X irradiation

Myogenic cells obtained from 12-day-old embryonic chicken hind limb and breast muscle were exposed to 5000 rads of X irradiation. Although 10% of the initial cell dissociates were killed by irradiation, the remaining cells were comparable to controls in plating efficiency and light microscopic morphology. Moreover, there was no increase or loss of cells for at least 72 hr in vitro when plated at a density of 2 × 10⁶ cells/60-mm plate. It was found that muscle cell fusion after irradiation proceeded at the same rate and to the same relative extent as in control cultures. Myotubes developed normally; cross-striations were prominent by 5 to 7 days of culture and the cells maintained a well-differentiated state for periods of at least 3 weeks in vitro. In control cultures continuously labeled with 1 μCi/ml of [³H]TdR, 75% of the nuclei within myotubes were heavily labeled by 118 hr; less than 15% of the nuclei within syncytia of irradiated cultures were labeled. Quantitative microphotometry of Feulgen-stained cultures demonstrated that all nuclei within control and irradiated myotubes contained the 2C complement of DNA. Similar experiments conducted with cells released from limbs and breasts of 10-day-old embryos revealed lower absolute levels of cytoplasmic fusion in both control and irradiated samples, however, there was slightly more cell death after exposure to X rays in 10-day-old than 12-day-old material. Nevertheless, considerable cell fusion occurred in irradiated limb and breast cell cultures, consistent with the conclusion that the commitment to myogenesis of prefusion myoblasts is extremely stable even in the face of massive ionizing radiation and that neither cell division nor replication of DNA is an obligatory prerequisite for the in vitro fusion and subsequent differentiation of skeletal muscle obtained from 10- and 12-day-old chick embryos.     

Whole body irradiation induces IFN-gamma production in BALB/c mice by preventing the appearance of a V alpha 14(+)NK T downregulatory population

Lymph node cells from TNCB-immune BALB/c mice fail to produce IFN-gamma when exposed to antigen in vitro. Conversely, lymph node cells of irradiated (550 rads) BALB/c mice produce IFN-gamma. Transfer experiments show that normal BALB/c mice contain cells which suppress IFN-gamma production. These downregulatory cells are CD4(+)alpha beta(+)and rearrange the invariant V alpha 14-J alpha 281 T cell receptor alpha chain, thus belonging to the NK T cell subset. Downregulatory cells probably act by producing IL-4 as their effect is blocked by mAb to IL-4.     

Meiotic maternal chromosomes introduced to the late mouse zygote are recruited to later embryonic divisions

The aim of this study was to investigate the fate of an additional female genome introduced to a dividing zygote. Maternal chromatin in the form of karyoplasts containing a metaphase II spindle were fused to zygotes blocked in anaphase or telophase of the first cleavage. Permanent preparations made 20-40 min after fusion at anaphase revealed that the donor maternal chromosomes had entered anaphase or telophase in 16 out of 18 cases. A further two groups of embryos that were fused at either anaphase or anaphase/telophase were cultured to the first division. Division occurred 50 min after fusion in both groups of embryos (86 and 85.1%, respectively), of which most divided to two cells (80 and 71.6% of total) and the remainder divided to three cells. About two thirds of two-cell embryos contained an extra nucleus in one blastomere. Nuclei containing donor maternal chromosomes reached a similar size to recipient nuclei in 68% of embryos derived from anaphase-blocked zygotes, in contrast to 31.1% of embryos derived from anaphase/telophase-blocked embryos. Replication of DNA in donor nuclei closely followed the timing and intensity of that in control embryos. When fixed 24 hr after fusion, one third of embryos were still at the two-cell stage, with one or both blastomeres showing a single metaphase plate of the second cleavage. In the remaining embryos, three or four cells were present, some containing two nuclei. Blastocysts developed in 50% of fused embryos and three young were born after transfer of cleaving hybrid embryos to recipients. Chromosome preparations from bone marrow of the young contained 3-4 tetraploid metaphase plates per several hundred plates counted compared with none in control embryos. In conclusion, additional maternal chromosomes can be introduced at the late-dividing zygote and join the embryonic cell cycles during subsequent divisions. This method may provide a useful approach for studying changes specific to the maternal genome during early cell cycles of the mammalian embryo.     

Association of endogenous retroviruses with radiation-induced leukemias of BALB/c mice.

X-irradiation of BALB/c mice in the second month of life induced a high incidence of generalized lymphatic leukemia of T-cell origin, beginning at 7 months of age. Infectious ecotropic murine leukemia virus (B-tropic predominant over N-tropic) was isolable from all tumor extracts but exhibited a wide titer range among individual leukemias. Detection of infectious xenotropic virus usually required extensive amplification on indicator cells. Dual-tropic (mink cell focus-forming) virus has not been found in the leukemias. Expression of ecotropic virus in tail extracts prepared at 6.5 months of age, although greatly enhanced compared with unirradiated controls, was not found to be prognostic of tumor development in individual mice. We conclude that leukemogenesis does not show a simple dependence on infectious murine leukemia virus expression in these mice.     

Overexpression of insulin-like growth factor (IGF)-I receptor enhances inhibition of DNA replication in mouse cells exposed to x-rays

Previous studies from our laboratory provided evidence for the operation of signal transduction pathways involving ras, myc, and staurosporine-sensitive protein kinases in the regulation of DNA replication in irradiated cells. Because ras and myc are also involved in the signal transduction elicited in response to ligand activation of growth factor receptors, we wondered whether growth factor receptors are upstream elements in the regulation of DNA replication in irradiated cells. Here, we report on the role of insulin-like growth factor I receptor (IGF-IR) in the regulation of DNA replication in irradiated cells. We compare radiation-induced inhibition of DNA replication in BALB/c 3T3 cells with that in P6 cells. P6 cells are derived from BALB/c 3T3 cells by transfection with a vector expressing IGF-IR, leading to 30-fold overexpression. We observe a significantly stronger inhibition of DNA replication after irradiation in P6 as compared with BALB/c 3T3 cells at all doses examined. Sedimentation in alkaline sucrose gradients shows that the increased inhibition in P6 cells is due to an increased inhibition of replicon initiation, the main controlling event in DNA replication. Staurosporine at 20 nM reduces radiation-induced inhibition of DNA replication in BALB/c 3T3 cells, but has only a small effect in P6 cells. Caffeine at a concentration of 1 mM, on the other hand, removes over 60% of the inhibition in both cell lines. The results implicate IGF-IR in the regulation of DNA replication in irradiated cells, but also suggest differences between cells of different origins in the proteins involved in the regulating signal transduction pathway.     

Radiation induced bystander signals are independent of DNA damage and DNA repair capacity of the irradiated cells

Evidence is accumulating that irradiated cells produce signals, which interact with non-exposed cells in the same population. Here, we analysed the mechanism for bystander signal arising in wild-type CHO cells and repair deficient varients, focussing on the relationship between DNA repair capacity and bystander signal arising in irradiated cells. In order to investigate the bystander effect, we carried out medium transfer experiments after X-irradiation where micronuclei were scored in non-targeted DSB repair deficient xrs5 cells. When conditioned medium from irradiated cells was transferred to unirradiated xrs5 cells, the level of induction was independent of whether the medium came from irradiated wild-type, ssb or dsb repair deficient cells. This result suggests that the activation of a bystander signal is independent of the DNA repair capacity of the irradiated cells. Also, pre-treatment of the irradiated cells with 0.5% DMSO, which suppresses micronuclei induction in CHO but not in xrs5 cells, suppressed bystander effects completely in both conditioned media, suggesting that DMSO is effective for suppression of bystander signal arising independently of DNA damage in irradiated cells. Overall the work presented here adds to the understanding that it is the repair phenotype of the cells receiving bystander signals, which determines overall response rather than that of the cell producing the bystander signal.     

Time-lapse analysis and normal stages of development of cleavage and blastocyst formation in the marsupials the brown antechinus and the stripe-faced dunnart

The first six normal stages of cleavage and blastocyst formation in vitro of the brown antechinus and the stripe-faced dunnart are described based on 429 embryos from 37 dunnarts and 143 embryos from 32 antechinus and an information from previous studies. Embryos were cultured in Dulbecco's modified Eagle's medium with high glucose and 10% foetal calf serum at 35 degrees C and 5% CO2 in air. Seven antechinus and 18 dunnart embryos were cultured in Rose chambers and filmed by time-lapse cinematography, with an exposure of 0.2 sec and at a rate of 1 frame/30 sec. A rim of zona was formed around the yolk mass during early cleavage. Blastomeres attached to this rim, ensuring that the zona in the hemisphere containing the yolk mass was the first to be lined by blastomeres during blastocyst formation, which is completed at about the 32-cell stage. Two foci were established during cleavage. The descendants of the first cell to divide at the four-cell stage included, at both the fourth and fifth division, the first cell to divide and the cell with the shortest cell cycle time and had the shortest average cell cycle time at each division. The descendants of the cell opposite the first cell to divide at the four-cell stage included, at both the fourth and fifth divisions, the last cell to divide and the cell with the longest cell cycle time and had the longest average cell cycle time at each division.     

Evolution of fast development of planktonic embryos to early swimming

Planktonic embryos of marine animals swim at an early stage and age. Although natural selection has apparently favored rapid development of structures for swimming, taxa have not converged on the same, minimal time from first cell division to first swimming. Comparisons of 34 species with planktonic embryos in 10 phyla revealed factors that account for variation in time to swimming. Time to first swimming correlated significantly with time from first to second cleavage (first cell cycle) in analyses of all embryos sampled and separately within the Spiralia and Echinodermata. Time to first swimming also correlated significantly with egg diameter in some clades, but not in all. Correlations between egg diameter and cell cycle duration were low except for the three species of Urochordata. Development to a feeding or nonfeeding larva did not affect time to first swimming beyond effects attributable to egg size. Time to first swimming did not correlate with type of locomotion developed (uniciliated cells, multiciliated cells, or muscle). Nonetheless, differences in locomotion are associated with changes in cell cycle durations prior to swimming. The ratios of time to first swimming and time for first cell cycle suggests that allocation of time to multiplication of cells versus differentiation of cells is resolved differently in species with different types of locomotion.     

The role of calcium in DNA synthesis and development of mouse preimplantation embryos in vitro.

The effect of calcium upon embryonic growth was studied using cultured mouse preimplantation embryos. Both morphological development of the embryos and embryo DNA synthesis were shown to be dependent on the Ca2+ concentration in the medium in which the embryos were grown. Reduction of the calcium concentration below 10(-5) M completely blocked cell division and blastocyst formation in the cultured embryos, but only moderately inhibited embryo DNA synthesis. Trifluoperazine, a calmodulin antagonist, strongly inhibited the Ca(2+)-dependent DNA synthesis in the embryos. On the other hand, the drug only slightly affected the morphological development of the embryos. These results demonstrate that calcium independently affects two different aspects of the embryo development, i.e. DNA synthesis and cell division. It is suggested that the former effect is calmodulin-dependent, while the latter involves the calcium-dependence of metabolite transport through the cell membranes.     

Regulation of the anti-alpha (1,3) dextran response: two populations of dextran-reactive B cells that differ in their T cell requirements for induction to antibody synthesis

The in vitro antibody response to dextran B1355S, a thymus-independent Type 2 antigen, requires T cell-derived lymphokines but is not thought to require an activation signal from an antigen-specific T helper cell. The present study demonstrates that there are two dextran-reactive B cell populations in BALB/c mice with respect to the T cell requirements for the generation of antibody-forming cells. One population found among dextran-reactive spleen B cells from 12- to 14-mo-old BALB/c mice generated anti-dextran PFC in the presence of B cell growth factor (BCGF II) and IL 2 or the combination of BCGF II, IL 2, and IFN-gamma. A second population of dextran-reactive B cells found in spleen and Peyer's patches of 2-mo-old unprimed mice did not respond to these same lymphokines, but did generate anti-dextran plaque-forming cells in the presence of Thy-1.2+, L3T4+ T cells from Peyer's patches. However, splenic B cells obtained from 2-mo-old mice that had been primed with dextran 2 to 3 days after birth were shown to be responsive to the same lymphokines as dextran-reactive B cells from 12- to 14-mo-old mice. These results suggest that previous priming with dextran B1355S induces a dextran-specific B cell population that can be activated to antibody-forming cells in the presence of antigen and T cell-derived lymphokines, whereas a second, unprimed population requires an additional activation signal from L3T4+ T cells.