Number Of Cells In Day Research Articles

28 SIMPLIFIED ACTIVATION METHOD TO IMPROVE THE IN VITRO DEVELOPMENT OF HANDMADE CLONED (HMC) PORCINE EMBRYOS

Delayed activation is commonly used in pig somatic cell nuclear transfer (SCNT) where electrical activation is followed by chemical activation. However, chemical incubation of several hours (up to 4 or 6) is logistically not very convenient even though handmade cloning (HMC) could improve the overall efficiency of pig cloning (Du et al. 2007 Theriogenology 68, 1104–1110). It was reported that a brief exposure of cycloheximide (CX) before electrical activation could significantly increase developmental rate and total blastocyst cell number when simultaneous activation was performed in micromanipulator-based pig cloning (Naruse et al. 2007 Theriogenology 68, 709–716). The purpose of our present work is to investigate whether such activation method is also applicable for pig HMC. Data were analyzed by t-test using SPSS (11.0, SPSS Inc., Chicago, IL, USA). After 42 h in vitro maturation, cumulus cells were removed. In vitro-cultured porcine fetal fibroblasts were used as donor cells. Cytoplast-fibroblast pairing, electrical fusion and activation of fused cytoplast-fibroblast pairs were performed as described previously (Kragh et al. 2005 Theriogenology 64, 1536–1545; Du et al. 2005 Cloning Stem Cells 7, 199–205). Three groups were compared due to different activation protocol. In Group 1 (control), reconstructed embryos were cultured in porcine zygote medium 3 (PZM3) supplemented with 4 mg mL–1 BSA, 5 μg mL–1 cytochalasin B (CB), and 10 μg mL–1 CX for 4 h. In Group 2 (CX priming), fused pairs and the other halves of cytoplasts were incubated in HEPES-buffered TCM-199 medium supplemented with 10% calf serum, 10 μg mL–1 CX for 10 min just before the second fusion or electrical activation. In Group 3 (CB + CX priming), treatment similar to Group 2 was performed except that additional 5 μg mL–1 CB was added for the 10-min incubation. Reconstructed embryos were in vitro cultured in the well of the well (WOW) system for 6 days. Blastocyst rates and total cell numbers of Day 6 blastocysts were evaluated. As illustrated in Table 1, embryos pretreated with both CB and CX gave the best results, with better blastocyst formation (53.8 ± 4.8%; mean ± SEM) and higher cell number (77.2 ± 5.4) compared to the other 2 groups. Our data suggested that CX and CB priming could be used as a solution to the long chemical incubation in porcine SCNT by HMC, making the embryos more receptive to electrical activation. Table 1.In vitro development of HMC reconstructed embryos with different activation protocols

194 ANTIOXIDATIVE EFFECT OF BROWN ALGAE PHLOROTANNINS ON REACTIVE OXYGEN SPECIES GENERATION AND EMBRYO DEVELOPMENT OF PORCINE PARTHENOGENETIC EMBRYOS UNDER OXIDATIVE AND HEAT-STRESSED CONDITIONS

We investigated the antioxidative effect of brown algae phlorotannins on reactive oxygen species (ROS) generation and embryo development of parthenogenetically activated porcine embryos under oxidative and heat-stressed conditions. Cumulus–oocyte complexes (COCs) were aspirated from follicles on the surface of porcine ovaries collected from an abbattoir. COCs were matured in NCSU-23 containing 10% (v/v) porcine follicular fluid and hCG during the first 22 h, followed by an extra 22 h of culture in hormone-free NCSU-23. After 44 h of maturation, oocytes were denuded of cumulus cells and used for parthenogenetic activation. Oocytes were activated by single 100-�s pulse of 1.5 kV cm-1 DC in 1-mm electrodes. Activated oocytes were cultured for 5 h in NCSU-23 containing BSA, EGF, and 5 �g mL-1 cytochalasin B. Embryos were then cultured for 7 days in PZM-5 medium that was a slightly modified version of the PZM-4 medium reported by Yoshioka et al. (2002 Biol. Reprod. 60, 112–119). In Experiment 1, after parthenogenetic activation, embryos were cultured for 7 days at 38.5�C under 5% O2, 5% CO 2, and 90% N2 (defined as 5% O2) as a control. Embryos were also cultured under 5% CO2 in air (defined as 20% O2) with or without 100 ng mL-1 brown algae phlorotannins extracted from Ecklonia kurome. The number of embryos developed to the blastocyst stage was observed on Day 6. The total cell number of Day 7 blastocysts was counted by DAPI staining of nuclei. On Day 2, intracellular ROS levels of individual embryos were measured with fluorescent dyes (222,722-dichlorodihydrofluorescein diacetate). In Experiment 2, on Day 1 or 2, embryos cultured in 5% O2 concentration at 38.5�C were exposed to 41.5�C for 6 h with or without 100 ng mL-1 phlorotannins and cultured at 38.5�C until Day 7. After 6 h of heat-shock on Day 1 or Day 2, intracellular ROS levels were measured as described in Experiment 1. Statistical analysis was carried out by ANOVA. In Experiment 1, the rate of blastocyst formation and the total cell number were significantly decreased (P < 0.05) when embryos were cultured under 20% O2 compared to 5% O2. In contrast, addition of phlorotannins significantly increased the rate of blastocyst formation under high O2 concentration. ROS levels were also significantly increased by higher O2 concentration. In contrast, addition of phlorotannins significantly reduced the ROS levels. In Experiment 2, heat-shock to embryos on Days 1 and 2 significantly (P < 0.05) decreased the rate of blastocyst formation compared to the control. In contrast, addition of phlorotannins significantly (P < 0.05) increased embryo development and decreased the intracellular ROS levels of heat-stressed embryos. These results indicate that oxidative and heat stress conditions decrease embryo development and increase the level of intracellular ROS. However, addition of phlorotannins promotes embryo development by decreasing the oxidative stress.

Effects of transient PTH on early proliferation, apoptosis, and subsequent differentiation of osteoblast in primary osteoblast cultures

In primary calvarial osteoblast cultures derived from transgenic mice expressing green fluorescent protein (GFP) under the control of 3.6-kb Col1a1 promoter, the emergence of GFP signal marks the transition of multipotential osteoprogenitors into preosteoblasts. Early transient treatment (days 1-7) of these cultures with parathyroid hormone (PTH) has an anabolic effect that is not associated with an increase in total DNA content or cell number in day 21 cultures. In the present study, the effect of early PTH treatment on cell proliferation and apoptosis was examined in greater detail in GFP(+) and GFP(-) cells using flow cytometry. In preconfluent cultures, PTH significantly reduced the proportion of cells in S phase but increased those in G(0)/G(1) and G(2)+M phases in both GFP(+) and GFP(-) subpopulations. PTH decreased apoptosis only in GFP(-) but not GFP(+) cells, indicating an increased survival of GFP(-) cells. In contrast, PTH did not change the amounts of cell proliferation and apoptosis seen in either compartment after these cultures reached confluence. To further assess the effect of early PTH treatment on osteogenic differentiation, secondary cultures of sorted GFP(+) or GFP(-) cells were obtained from day 7 primary cultures that had been treated for 1 wk with PTH. This treatment resulted in larger areas of GFP expression accompanied by increased xylenol orange/von Kossa staining in the secondary cultures of GFP fractions. Early transient PTH treatment appears to enhance the commitment of progenitor cells to an osteogenic fate and results in a higher proportion of cells that achieve full osteoblast differentiation.

Higher survival rate of vitrified and thawed in vitro produced bovine blastocysts following culture in defined medium supplemented with β-mercaptoethanol

The present study was conducted to compare bovine embryo developmental quality, after culture in different defined culture media, up to blastocyst stage, and subsequently cultured in media supplemented with beta-mercaptoethanol (β-ME) following blastocyst vitrification and thawing. In part one of this study, presumptive zygotes were randomly allocated into the following media: (1) CR1, (2) KSOM, (3) SOF, and (4) sequential KSOM-SOF. In the second part of the study, blastocysts derived from four different culture media were subjected to a solid surface vitrification (35% (v/v) ethylene glycol + 0.5 M Sucrose + 5% (w/v) Polyvinylpyrrolidone (PVP), and tested for the effect of β-ME on their post-vitrification survival. Following thawing, blastocysts were cultured with or without β-ME. Culture medium had no effect on cleavage rates; however, a significantly greater number of zygotes cultured in KSOM, KSOM-SOF, or SOF developed to the 8-cell stage, compared with those cultured in CR1. A greater proportion of the zygotes cultured in SOF or KSOM-SOF reached blastocysts, than did those cultured in CR1 or KSOM. The use of sequential KSOM-SOF significantly increased total cell numbers of Day 7 expanded-blastocysts when compared to those cultured in CR1, KSOM, or SOF. Addition of β-ME into culture media after vitrification and thawing improved blastocyst survival, hatching rates, and total cell numbers of blastocysts. In conclusion, supplementation of β-ME into culture medium after vitrification and thawing significantly increased blastocyst survival, hatching rates, and their total cell numbers. These results suggest that vitrified IVF embryos should be thawed and briefly cultured in β-ME medium prior to embryo transfer.

63 IMPROVED IN VITRO DEVELOPMENT OF PORCINE EMBRYOS PRODUCED BY NUCLEAR TRANSFER, IVF AND PARTHENOGENESIS

Porcine nuclear transfer (NT) is an inefficient process and it is necessary to use as many as 120 NT embryos for each foster mother to obtain small litters of live piglets. In these experiments, we evaluated the effects of culture atmosphere and medium on the development of NT embryos by monitoring blastocyst rate and cell number of Day 6 blastocysts. Age matched IVF and parthenogenetic embryos were also evaluated for comparison. For all experiments a pool of oocytes was aspirated from ovaries collected in a local abattoir. Following aspiration, oocytes were allowed to mature for 40 h in North Carolina State University (NCSU)-37 medium (supplemented with cAMP and hCG/eCG for the first 22 h). After removal of the cumulus cells, denuded oocytes with polar bodies were selected for NT, enucleated, fused with fetal fibroblasts, and sequentially activated electrically and chemically by 3 h of treatment with 6-dimethylaminopurine (6-DMAP). A second group of oocytes from the same denuded pool were maintained in TL-HEPES medium and activated in parallel with the NT group to produce parthenogenetic embryos. A third group was fertilized with frozen-thawed epididymal semen and co-cultured for ∼12 h to give IVF embryos. All three treatment groups were subdivided into a control subgroup and an experimental subgroup. In the first experiment, we compared the effects of atmosphere (20% vs. 5% oxygen) on in vitro embryonic development in NCSU-23 medium. In the second experiment, we used only the 5% oxygen concentration and compared different culture media. One subgroup was maintained in standard NCSU-23 medium and the second subgroup was cultured in a two-step system for the first 58 h in modified NCSU-23 (without glucose but supplemented with 2.0 mM lactate and 0.2 mM pyruvate), followed by addition of glucose to give a final concentration of 5.55 mM. Data were statistically analyzed by analysis of variance and chi square test. Blastocyst rate and mean cell number in all three embryo groups were improved under 5% oxygen. The most dramatic effect was observed in the NT group, in which the blastocyst rate increased significantly (P < 0.001) from 6.7% ± 5.9 (n = 279) to 19.6% ± 8.9 (n = 250) and mean cell number increased from 17.7 ± 12.1 to 25.8 ± 10.3 cells per blastocyst. With 5% oxygen there was also an increase of blastocyst rates and mean cell numbers in both IVF and parthenogenetic groups. In the second experiment, blastocyst rate for NT embryos increased significantly (P < 0.05) from 21.8% ± 7.6 (n = 242) in conventional NCSU-23 to 31.5% ± 11.0 (n = 271) in the modified system whereas there was almost no difference in the mean cell number of both groups (29.2 ± 4.3 vs. 31.5 ± 5.3). In the groups of IVF and parthenogenetic embryos no difference was found. These results indicate that both the reduced oxygen and the modified culture medium are important for pre-implantation development of porcine nuclear transfer embryos.

155 QUALITY OF PORCINE EMBRYO PRODUCED IN TWO EMBRYO CULTURE MEDIA AS ASSESSED BY TOTAL CELL NUMBER AND TIME COURSE OF BLASTOCYST HATCHING

The ability to produce porcine embryos of good quality will have a significant impact on a number of porcine assisted reproductive technologies, such as cloning, intracytoplasmic sperm injection, and embryo cryopreservation. However, porcine embryos resulting from current serum-free embryo culture systems differ significantly both structurally and functionally from those derived in vivo (Wang et al. 1999 Mol. Reprod. Dev. 53, 99-107). In this experiment, the quality of porcine embryos produced by North Carolina State University (NCSU)-23 medium (Petters and Wells 1993 J. Reprod. Fertil. Suppl. 1993, 48, 61-73) and porcine zygote medium (PZM)-1 (Yoshioka et al. 2002 Biol. Reprod. 66, 112-119) were compared by assessing the total cell number and the time course of in vitro blastocyst hatching. Porcine embryos were produced by in vitro maturation and fertilization using serum-free systems. After fertilization, presumptive zygotes were randomly allocated to either PZM-1 or NCSU-23 for subsequent development. On Day 4 of culture, the embryo culture media were supplemented with 10% fetal bovine serum (FBS). Day 6 blastocysts from each group were counted and the blastocysts were subsequently fixed in 4% formalin for counting the total cell number. The cell number in each embryo was determined by counting the nuclei after staining with bisbenzimide (Hoechst 33342). To assess the hatching ability of blastocysts, Day 6 blastocysts were cultured until Day 9 and hatched blastocysts were counted daily. Day 6 blastocyst rates (ratio of blastocysts to oocytes) and total cell number count were replicated three times. The time course of blastocyst hatching experiment was repeated four times. The data were analyzed using a chi-square test, Fisher's exact test, or Student's t-test. The blastocyst rate from culture in PZM-3 was 19.4 � 0.96% (mean � SEM), which was similar to that (16.7 � 3.2%) resulting from culture in NCSU-23 (P > 0.05). However, the total cell number in Day 6 blastocysts cultured in PZM-3 was significantly higher than for blastocysts cultured in NCSU-23 (57 � 3.1 vs. 46 � 1.7; P < 0.01). The total hatching rates (ratio of hatched blastocysts to total blastocysts) by Day 9 were similar between the two culture systems (50.1 � 9.1% vs. 50.7 � 4.1%; P > 0.05). However, on Day 6, 2.1% of blastocysts from PZM-3 culture hatched whereas no blastocysts from NCSU-23 culture hatched. The cumulative hatching rates from PZM-3 culture on Day 7 were significantly higher than those from NCSU-23 culture (15.1 � 3.8% vs. 2.6 � 1.1%; P < 0.01). In conclusion, these data suggest that blastocysts produced in PZM-3 medium have better quality than blastocysts produced in the NCSU-23 culture system as assessed by the total cell number and the time course of blastocyst hatching. This project was supported by a grant from the National Institutes of Health (U42 RR 018877).

Evaluation of different culture systems on the in vitro production of bovine embryos

The objective of this study was to determine the development potential and quality of in vitro produced bovine embryos cultured individually or in groups. After IVM and IVF, presumptive zygotes were cultured in groups or individually, either in drops or in the modified “well of the well” (mWOW) system. In Experiment 1, four culture systems were utilized: T1: drop in group (control); T2: mWOW in groups; T3: mWOW individually; and T4: drop individually. Cleavage and blastocyst rates at Days 6, 7 and 8 and total cell number of Day 6 blastocysts were similar ( P > 0.05) for all treatments. However, in Day 7 blastocysts, total cell number was lower ( P < 0.05) in embryos cultured individually in a small drop than those cultured in the mWOW. In Experiment 2, blastocysts of T1, T2 and T3 were allocated into two groups, control and vitrified. After warming, the vitrified embryos were cultured for 72 h. At 48 h, the development of the Days 6 and 7 embryos was similar ( P > 0.05) for all treatments in the control group. For the vitrified embryos, lower hatching rates ( P < 0.05) were observed in the T3 group. In conclusion, embryos cultured in groups in the mWOW system had the same blastocyst rates but better quality (measured by their survival after vitrification) than those cultured individually in the mWOW system.

Effect of supplementation of leukemia inhibitory factor and epidermal growth factor on murine embryonic development in vitro, implantation, and outcome of offspring

Objective To evaluate the supplementation of leukemia inhibitory factor (LIF) or epidermal growth factor (EGF) in a single culture system (IVF50) and a sequential culture system (IVF50-S2) to determine whether they had any additional benefit on embryo development and implantation. Design Prospective controlled animal study. Setting University research laboratory. Subject(s) Two-cell embryos from F1(CBA × C57BL) mice. Intervention(s) Embryos were randomly cultured in different growth factor-treated or untreated in vitro systems. Main outcome measure(s) Blastocyst formation and morphology, total cell numbers of day 5 blastocysts, birth rates, and characteristics of the offspring. Result(s) The beneficial effects of LIF or EGF on blastocyst development and morphology were observed only in IVF50 medium but not in sequential IVF50-S2 media. In addition, blastocysts generated from the LIF-supplemented cultures had higher total cell numbers and higher total birth rates after transfer compared to those from IVF50 medium alone. No significant effect on fetal development was observed but pups born after treatment in LIF-supplemented sequential cultures had prolonged gestation and increased birth weights. Conclusion(s) The beneficial effects of LIF and EGF on in vitro blastocyst development are mainly seen under suboptimal culture conditions (simple medium only) and their effects are masked in an improved in vitro system (sequential culture media). Supplementation of culture media with such factors, however, is still an attractive approach in enhancing embryo viability through increased total cell numbers. The beneficial effect of LIF in improving implantation and subsequent increased birth rates should be further explored.

Cellular and molecular deviations in bovine in vitro-produced embryos are related to the large offspring syndrome.

The large offspring syndrome (LOS) is observed in bovine and ovine offspring following transfer of in vitro-produced (IVP) or cloned embryos and is characterized by a multitude of pathologic changes, of which extended gestation length and increased birthweight are predominant features. In the present study, we used bovine blastocysts to analyze cellular parameters, i.e., the number of cells in Day 7 blastocysts and the size of Day 12 elongating blastocysts, and molecular parameters, i.e., the relative abundance of developmentally important genes: glucose transporter (Glut) 1, Glut-2, Glut-3, Glut-4, heat shock protein (Hsp) 70.1, Cu/Zn-superoxide dismutase (SOD), histone H4.1, basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF) I receptor (R), and IGFII-R. Some blastocysts were produced by in vitro maturation and fertilization followed by in vitro culture in synthetic oviduct fluid medium supplemented with BSA or human serum or by in vivo culture in the sheep oviduct. Other blastocysts were derived in vivo from the uterine horns of superovulated donors. The findings made in the early embryos were related to a representative number of calves obtained from each production system and from artificial insemination (AI). In vitro culture of bovine embryos in the presence of high concentrations of serum or BSA significantly increased the number of cells in Day 7 blastocysts, the size of blastocysts on Day 12, and the relative abundance of the transcripts for Hsp70.1, Cu/Zn-SOD, Glut-3, Glut-4, bFGF, and IGFI-R when compared with embryos from the in vivo production groups. Birthweights of calves derived from IVP embryos were significantly higher than those of calves derived from sheep oviduct culture, superovulation, or AI. The results support the hypothesis that persistence of early deviations in development is causally involved in the incidence of LOS, in particular in increased birthweights. The cellular and molecular parameters analyzed in this study can be considered early markers of LOS in cattle.

Comparison of the effects of using standard and simple portable CO 2 incubators on the in vitro developmental competence of bovine embryos reconstituted by somatic cell nuclear transfer

In an attempt to determine the cultural factors that would improve cloning efficiency, we compared the effects of two incubation systems—a simple portable system and a standard CO 2 incubator—on the production of bovine embryos by electrofusion of quiescent fetal fibroblast nuclei to enucleated oocytes matured in vitro. While the temperature (38.5 °C) and CO 2 concentration (5%) were similar in both systems, the portable incubator operated in a vacuum of 300 mmHg and at an O 2 level of 8–10%, which is lower than the standard. Although there were no significant differences between the two systems in terms of in vitro oocyte maturation (MII stage), fusion rates, and the number of cells in Day 7 blastocysts, significantly higher proportions of nuclear-transferred oocytes cleaved ( P<0.05) and developed to the blastocyst stage ( P<0.01) in the portable incubator (70.5±0.6 and 36.1±1.4%, respectively) than in the standard incubator (64.1±3.2 and 23.5±1.4%, respectively). Following the transfer of six blastocysts from the portable incubator group to three recipients, survival rates on Days 60, 90, and 120 were 100, 66.7 and 33.3%, respectively. This relatively high early embryonic loss may be associated with multiple pregnancy complications or other abnormalities of placentation frequently observed in cloned embryos. Further studies using this portable incubator system are needed to determine the optimum levels of O 2, CO 2, and air pressure.

Routine addition of human insulin-like growth factor-I ligand could benefit clinical in-vitro fertilization culture.

Animal studies suggest that the insulin-like growth factors play an important role in preimplantation embryo development. Human preimplantation embryos express mRNA for insulin-like growth factor-I receptor (IGF-IR) but the ligand, insulin-like growth factor-I (IGF-I), is not expressed by the embryo until after implantation. We tested the hypothesis that IGF-I produced by the female reproductive tract may bind to these receptors, augmenting embryo survival, growth and development. Transcripts of mRNA for IGF1 were detected using reverse transcription-polymerase chain reaction in midcycle human Fallopian tube. Immunohistochemistry localized immunoreactive IGF-I to the cytoplasm of all the major structures of the Fallopian tube, with the most intense staining seen in the tubal epithelial lining. Maternally produced IGF-I was present in the fluid found in the human tube and uterus at concentrations of 8.0 and 10.9 nM respectively. Supplementation of culture medium with IGF-I increased the proportion of embryos developing to the blastocyst stage from 35% in controls to 60% in the presence of IGF-I. In addition the total number of cells in day 6 blastocysts was increased by 19% (64.44 versus 54.08% in control, not significant), due entirely to a statistically significant 59% increase (25 versus 15.75%, P = 0.020) in the number of cells in the inner cell mass. The effect of IGF-I was mediated through the IGF-I receptor. Immunocytochemistry using an alphaIR3 antibody confirmed the presence of IGF-I receptor in human blastocysts and the same antibody completely inhibited the stimulation of blastocyst formation by IGF-I. These data suggest that human preimplantation development is enhanced by maternal IGF-I. Mimicking this in-vivo paracrine relationship may improve clinical in-vitro embryo culture and IVF pregnancy rates.

Viability of bovine blastocysts obtained after 7, 8 or 9 days of culture in vitro following vitrification and one-step rehydration

This study examined morphological appearance, viability and hatching rates in relation to the total cell number following vitrification of in vitro produced bovine blastocysts and expanded blastocysts. In Experiment 1, embryos obtained after 7, 8 or 9 d of culture were pooled and equilibrated in either 10% ethylene glycol (EG) or 10% EG plus 0.3M trehalose in Dulbecco's phosphate buffered saline (DPBS) supplemented with 10% calf serum and 0.6% BSA for 5 min each, at room temperature, and then vitrified together in precooled vitrification solutions consisting of 40% EG (Treatment 1), 40% EG plus 0.3M trehalose (Treatment 2), 40% EG plus 0.3M trehalose and 20% polyvinylpyrrolidone (PVP, Treatment 3) in DPBS. The embryo viability and hatching rates of Treatment 1 (19 and 3%) differed significantly (P < 0.05) from those of Treatment 2 (56 and 31%) and Treatment 3 (70 and 43%). There was a significant difference (P < 0.05) in embryo viability between Treatment 2 (31%) and Treatment 3 (43%). In Experiment 2, Day 7, 8 and 9 embryos were vitrified separately, with higher viability and hatching rates in Experiment 1 than in Experiment 2. The viabilities of Day 7 (87%), 8 (71%) and 9 (46%) embryos differed significantly (P < 0.05). Again, there were significant differences (P < 0.01) among the hatching rates of Day 7 (75%), 8 (38%) and 9 (9%) embryos. The total cell number of hatched blastocysts was then determined by differential fluorochrome staining. The total cell number of Day 7, 8 and 9 embryos differed significantly (P < 0.05).

In Vitro Development of Rabbit Pronuclear Embryos in Rabbit Peritoneal Fluid1

The use of rabbit peritoneal fluid (PF) for the culture of rabbit embryos in vitro was evaluated. Development of zygotes cultured in PF and Earle's balanced salts solution (EBSS) + 10% fetal calf serum (EBSS/FCS) was compared. The effects of increasing the concentration of PF in EBSS and of culturing embryos in fractionated PF were also investigated. In addition, embryonic development in PF was compared to that in vivo. Development to hatching blastocysts was enhanced with PF (73%) compared to EBSS/FCS (3%, p less than 0.001). PF manifested greater mitogenic activity than EBSS/FCS, as indicated by higher cell number in embryos at 48, 72, and 96 h post-mating/hCG (p less than 0.001). PF also promoted blastocyst cell proliferation in a dose-dependent manner (r = 0.98, p less than 0.01); however, embryo growth remained slower than in vivo. Culture in the high (greater than 30,000 Da) molecular mass fraction of PF reduced incidence of hatching (56% vs. 92%, p less than 0.001) and mean cell number in Day 4 blastocysts (151 +/- 4 vs. 243 +/- 5, p less than 0.001). Rates of blastocyst hatching (10%) and cell number (110 +/- 3) were further reduced in the low (less than 30,000 Da) molecular mass fraction. When the high molecular mass fraction was dialyzed, embryos did not develop beyond the early morula stage. This suggests that the interaction or the synergy of high and low molecular mass components of PF is necessary for optimum development of rabbit embryos.

Transfer of agammaglobulinemia in the chicken: I. Generation of suppressor activity by injection of bursa cells

Spleen cells from adult agammaglobulinemic (bursectomized) chickens taken 1 to 3 weeks after an injection of histocompatible bursa cells can inhibit the adoptive antibody response to B. abortus of normal spleen or bursa cells in irradiated recipients. Spleen cells from Aγ chickens not injected with bursa cells generally do not. Moreover, bursectomized chickens which have been reconstituted with spleen cells within the first week after hatching do not respond with suppressor cell formation upon bursa cell injection. This apparent “autoimmunization” with bursa cells induces suppressor T cells which are only minimally sensitive to treatment with mitomycin C or to 5000 R γ irradiation. The suppressor activity is neither induced nor potentiated by concanavalin A in vivo. It is much stronger in spleen than in thymus cells and appears to be macrophage independent and to require intact cells. The cell component which stimulates the suppressor activity is more pronounced on bursa than on spleen cells, and is at most present to a very limited extent on bone marrow, thymus, or peritoneal exudate cells. It is better represented in comparable cell numbers of Day 17 than of Day 14 embryonic bursa. The inducing cell component is present in the membrane fraction of disrupted bursa cells. Immunization with bursa cells from B locus histoincompatible chickens leads to suppressor activity against histocompatible bursa cells. Although the removal of Ig-bearing cells from bursa greatly diminishes its immunizing capacity, injection of serum IgM and IgG does not induce suppressor cells. It is suggested that tolerance to a B-cell antigen is lacking in adult Aγ chickens, resulting in an autoimmune response upon exposure to B cells. The B-cell antigen may be a cell surface-specific form of Ig, a complex of Ig and a membrane component, or a differentiation antigen which appears simultaneously with Ig during ontogeny.