Differentiation of Lens and Pigment Cells in Cultures of Brain Cells of Chick Embryos

Dissociated cells of brains (tel- and diencephalons) of 3.5-day-old chick embryos were cultivated in vitro under the cell culture conditions which are known to be permissive for neural retinal cells (NR cells) to transdifferentiate into lens and/or pigmented epithelial cells (PE cells). The differentiation of lentoid bodies (LBs) with lens-specific (δ-crystallin and PE cells with melanin granules was observed in such brain cultures.
LBs appeared in two different phases, i. e., 2–3 days and 16–30 days of cultivation, and after 40 days of culture these structures were formed in all 60 culture dishes. Sometimes, LBs were observed in foci of PE cells formed during earlier stages of brain cultures. When similar brain cultures were prepared with older embryos of 5-, 8.5-, 14-, and 16-days of incubation, no differentiation of lens and PE cells was observed.     

Effect of RU486 on different stages of mouse preimplantation embryos in vitro

17 beta-Hydroxy-11 beta(4-dimethylaminophenyl)-17 alpha-(1-propynyl)estra-4, 9-dien-3-one (RU486) inhibited the in vitro development of different stages of mouse preimplantation embryos under study. Two-celled embryos, morulae, and early blastocysts were obtained from B6D2F1 mice. The embryos were grown in Ham F-10 nutrient mixture (with glutamine) supplemented with sodium bicarbonate (2.1 g/L), calcium lactate (282 mg/L), and bovine serum albumin (fraction V, 3 mg/mL) at 37 degrees C in a humidified incubator supplied with 5% CO2 in air. RU486 was added to the culture medium at concentrations of 1, 5, 10, and 20 micrograms/mL. Culture medium with 0.05% ethanol served as the control. In vitro growth of embryos was assessed by the following criteria: (i) two-celled stage embryo development to blastocyst stage after 72 h, (ii) morula stage grown to blastocyst stage after 24 h, and (iii) early blastocyst stage development to hatching blastocyst after 12 h, in culture. RU486 inhibited the in vitro development of two-celled embryos, morulae, and early blastocysts at concentrations of 5, 10, and 20 micrograms/mL culture medium (p less than 0.001). The inhibitory effect of RU486 at these concentrations on the development of all the stages of embryos under study was irreversible. However, RU486 did not affect embryo development at 1 microgram/mL culture medium. The study indicates the direct adverse effect of RU486 at 5 micrograms/mL and higher concentrations in culture medium on the development of mouse preimplantation embryos in vitro, and it encourages its further investigation as a postcoital contraceptive in animal models and humans.     

Stage-related capacity for limb chondrogenesis in cell culture.

Cells from wing buds of varying-stage chick embryos were dissociated and grown in culture to test their capacity for cartilage differentiation. Micro-mass cultures were initiated with a cell layer greater than confluency, which occupied a restricted area of the culture dish surface (10–13 mm²). Cells from stage 24 chick embryo wing buds (prior to the appearance of cartilage in vivo) undergo cartilage differentiation in such cultures. Typically, during the first 1–2 days of culture, cells form aggregates (clusters of cells with a density 1.5 times greater than that of the surrounding nonaggregate area). By Day 3, virtually all aggregates differentiate into cartilage nodules which are easily recognized by their Alcian blue staining (pH 1.0) extracellular matrix. Subsequently, nodules increase in size, and adjacent nodules begin to coalesce. Micro-mass cultures were used to test the chondrogenic capacity of wing bud cells from chick embryos representing the different stages of limb development up to the appearance of cartilage in vivo (stages 17–25). Cells from embryo stages 21–24 form aggregates which differentiate into cartilage nodules in vitro with equal capacity (scored as number of nodules per culture). In contrast, cells from embryo stages 17–19 form aggregates in similar numbers, but these aggregates never differentiate into nodules under routine conditions. However, aggregates which form in cultures of stage 19 wing bud cells do differentiate into cartilage nodules if exposed to dibutyryl cyclic AMP and theophylline. Cells from stage 20 embryos manifest a varying capacity to form cartilage nodules; apparently, this is a transition stage. Cells from stage 25 embryos produce cartilage in vitro without forming either aggregates or nodules. Based on the results presented in this paper, the authors propose a model for cartilage differentiation from embryonic mesoderm cells involving: (1) aggregation, (2) acquisition of the ability to respond to the environment in the aggregate, (3) elevated intracellular cyclic AMP levels, and (4) stabilization and expression of cartilage phenotype.     

INDUCTION OF THE FROG HATCHING GLAND CELL FROM EXPLANTED PRESUMPTIVE ECTODERMAL TISSUE BY LICL

When cells of the superficial layer explanted from the presumptive ectoderm of a Rana japonica early gastrula embryo at stage 10 were cultured in standard salt solution for 4–7 days, they differentiated into cement gland cells (CGCs), cilia cells (CCs) and common epidermal cells (CECs). When, however, these explants were treated with LiCl and transferred to Barth's solution, hatching gland cells (HGCs) and pigment cells were induced.
The optimum condition for inducing differentiation of HGC was treatment with 70 mM LiCl for 6–8 hr at 18°C. The best ability to react to the HGC-inducing stimuli resided in the superficial layer of the dorsal presumptive epidermis of the embryo at stage 10. Upon repeated stimulation, explants from stage 8 embryos underwent differentiation into nerve and pigment cells, whereas those from stage 11 embryos differentiated into CCs and CECs. Under optimum conditions, the total volume of HGCs induced amounted to about 70% of the explanted tissue. The culture media from LiCl-induced HGCs showed an apparent jelly-digesting activity, strongly indicating that the cells were functionally identical with those differentiated in situ .     

Totipotency, somatic embryogenesis, and Harry Waris (1893–1973)

F. C. Steward and Jakob Reinert in the late 1950s, independently and with different degrees of scientific exactness, demonstrated that somatic cells of cultivated carrot can produce embryo-like structures in aseptic culture. Growth substances in the nutrient medium were viewed as central to the process. The now classic papers of Steward and Reinert have found a special and enduring place in the literature of plant development. But Harry Waris also deserves credit for his observation that vegetative cells sloughed off from aseptically germinated seedlings reared in liquid nutrient medium can produce 'embryos'. In his studies, seedlings of Oenanthe aquatica (Umbelliferae) were maintained in culture for protracted periods under nutrient conditions designed to foster imbalance in protein metabolism, but without exogenous growth hormones. Seedlings placed in media with high concentrations of glycine grew normally for 3–4 months; after this a "period of morbidity" occurred, followed by production of new plants from the root tips. These new plants, later called "neomorphs", in turn reproduced by colorless outgrowths of leaf epidermis. Such outgrowths, and "nodules" formed in a callus produced by the original seedlings, passed through stages described as "nodule", "fusiform", and a stage with two or more "lobes". Transfer of the neomorphs to a medium lacking glycine resulted in the development of normal plants. We show that Waris was among the first, if not the first, to observe and recognize somatic embryo production in aseptic culture, and indeed to call them "embryos". We also discuss his investigations in the context of understanding development at the cellular level, then and now.     

AGE-DEPENDENT CHANGE IN THE TRANSDIFFERENTIATION ABILITY OF CHICK NEURAL RETINA IN CELL CULTURE*

We examined how the transdifferentiation ability of neural retinal cells into lens and/or pigment cells in call culture is changed with the development of the donor. Cells dissociated from neural retinas of chick embryos ranging from 3-day-old to the stage immediately before hatching and of 3-day-old chicks were cultured for about 60 days. The results clearly indicated that the transdifferentiation ability decreased with age. The latest developmental stage at which the differentiation of lens cells took place was in 18-day-old embryos. A gradual decrease in this ability was shown by the comparison of crystallin content in cultures prepared from embryos at different stages. The differentiation of pigment cells was recognized in cultures of neural retinas earlier than in 15-day-old embryos. Such loss of the ability of neural retinal cells to transdifferentiate into pigment cells earlier than into lens cells can be partially attributed to inhibitory factors accumulated in medium conditioned with many neuronal cells present in cultures.     

Pluripotency of mouse embryonic cells on germline at 3.5–8.5 and 11.5 days post-coitum after aggregation with precompacted embryos

Albino mouse embryonic cells (Gpi-la/a) at 3.5–8.5 and 11.5 days were aggregated with zona cut 8–16 cell stage embryos from F₁ females (Gpi-1 b/b), respectively. The aggregated embryos were transferred to pseudopregnant female mice. The recipients were allowed to go to term or were dissected at mid-gestation to assess the donor contribution in the conceptuses using glucose phosphate isomerase (GPI) analysis. The donor cells, which were previously labeled with fluorescent latex microparticles, were aggregated with embryos, and the allocation of the donor cells at the compacted morula and blastocyst stages were observed under a fluorescence microscope. When 3.5 and 45 day old inner-cell-mass (ICM) cells were used, fertile chimeric mice were obtained (50 and 19%, respectively), and when 5.5 days old primitive ectoderm cells were aggregated, they did not form chimeras but contributed to the fetuses, placenta and membrane after 13.5 days of pregnancy. However, cells from further stages never contributed to the conceptuses even though they were analyzed after 10.5 days of pregnancy. The labeled donor cells at these stages were not positively incorporated in the interior part of the compacted morula and the ICM of the blastocyst stage unlike the ICM at 3.5 days post-coitum after overnight culture.     

Essential role of the yolk syncytial layer for the development of isolated blastoderms from medaka embryos

To investigate a possible role of the yolk syncytial layer (YSL) in the development of the medaka embryo, blastoderms were isolated at different stages of embryogenesis either with or without the layer and were incubated in a culture medium. The blastoderms from cleavage stage embryos (stage 8–9), in which the YSL had not yet formed, developed into an irregular mass of cells. But some of the blastoderms isolated with the YSL from the blastula embryos (stage 10) developed into embryo-like structures with apparent body axes and contained differentiated organs, such as the eye, ear, contractile heart, yolk sac-like sphere and posterior body trunk with notochord. The proportion of such explants increased as the developmental stage proceeded. However, the proportion was much smaller when blastoderms were isolated at the blastula stage without the YSL. These results suggest that the YSL is essential for the development of embryonic structures. At stage 12 (early gastrula), the frequency of formation of such structures was the same among blastoderms with or without the YSL, so that these embryos are apparently committed for pattern formation.     

Immunocytological study on the differentiation of chick and quail adenohypophysis epithelial rudiments, grafted or cultivated in vitro: evidence for polypeptidic hormones

Epithelial rudiments of adenohypohysis were removed from chick and quail embryos between days 3 and 5 of development. Chick rudiments were grafted for 11--13 days onto the chorioallantoic membrane of decapitated chick embryo hosts. Quail rudiments were cultivated in vitro for 6 days. Both grafted and cultivated Rathke's pouches differentiated into adenohypophyseal tissue. The adenohypophyseal tissue cultured on chorio-allantoic membrane exhibited cells reacting with the following immune sera: anti-beta-(1--24)ACTH, anti-alpha-(17--39)-ACTH, anti-alpha-endorphin, anti-beta-endorphin and anti-beta-LPH, which also gave a positive reaction when applied to adenohypophysis of corresponding age which had differentiated in situ. In situ, corticotrophs were located exclusively in the cephalic lobe of adenohypophysis. Therefore, the differentiation of corticotrophs in the whole graft, i.e., from both cephalic and caudal lobes of Rathke's pouch, showed that the cells of the caudal lobe, or at least some of them, were uncommitted when the rudiment was removed. In vitro, tissue derived from Rathke's pouch contained cells reacting with antibodies to beta-(1--24)-ACTH, alpha-(17--39)-ACTH, and beta-LPH, as did adenohypophysis from quail embryos of corresponding age (9--10 days), differentiated in situ. The differentiation of quail Rathke's pouch in vitro corroborates that differentiation can occur without influence from hypothalamus and, moreover, shows that at least some kinds of cells can differentiate without influence exerted by any other encephalic factors, and in the absence of mesenchyme. The question arises whether fibroblastic cells derived from Rathke's pouch cells act as feeder-cells and/or secrete some factors promoting differentiation.     

Increasing the rate of blastocyst formation and hatching from in vitro-produced bovine zygotes

This study was designed to identify parameters that would facilitate early selection of superior embryos, as well as to define culture conditions that could increase the proportion of embryos proceeding to the blastocyst stage. In the first experiment, the developmental potential of bovine embryos that had reached different stages of development after 60 h of culture following insemination was assessed. No 2-cell embryos underwent further cleavage. Of the 4-cell embryos (n = 188) only 12.2% progressed to the blastocyst stage, while 62.5% of 8-cell embryos (n = 480) did so (P < 0.01). In a further experiment, the effects of conditioning the culture medium (TCM 199) either with Buffalo rat liver cells (BRLC) or bovine oviductal epithelial cells (BOEC) and the effects of co-culture with either of these 2 cell types were examined. The percentage of 8-cell embryos proceeding to the morula and blastocyst stages was independent of cell type and culture system. However, BOEC-conditioned medium supported significantly lower production of blastocysts than any of the other culture methods. Only 24.1% of the former proceeded to the blastocyst stage after the full 10 d of culture, and only 3% hatched, values that were significantly lower than in the other 3 groups (P < 0.01). Among the latter, 44% progressed to the blastocyst stage in BRLC-conditioned medium while 44 and 46% reached that endpoint after co-culture with BOEC or BRL cells, respectively. The percentages that hatched among these were 28.2, 31 and 28.5%, respectively.     

Changes in the Endogenous Level and Effects of Abscisic Acid during Somatic Embryogenesis of Daucus carota L.

The amount of endogenous ABA in cell clusters and embryos remainedlow during the first 7 days of culture regardless of the presenceor absence of 2,4-D in the medium, but it increased toward the10th day of culture. The ABA content in developing embryos reacheda maximum on the 10th day then decreased. But, in cell clustersgrown in a medium containing 2,4-D (in which embryo formationand development were suppressed) the ABA content increased continuouslyuntil it reached the maximum value of 80 ng/g fr wt on the 13thday, then it decreased to a low level on the 17th day of culture. The effects of ABA on embryo formation and development wereinvestigated in a medium containing ABA at a concentration of100 ng/ml, a level comparable to the endogenous levels of ABAwhich we had measured. Due to continuous treatment with ABA,both the total number of embryos and the number of abnormalembryos (those with extremely elongated roots) decreased. Asimilar, but weaker effect, was obtained when ABA was appliedonly during the first 7 days of culture (embryo-forming stage).When ABA was applied during the last 7 days of culture (embryo-developingstage), its effect was marked especially on the suppressionof abnormal embryo formation. (Received July 18, 1981; Accepted September 17, 1981)     

Progression throughout All Stages of Meiosis from the Early Prophase of Newt Primary Spermatocytes in vitro

Stages of prophase of living primary spermatocytes were determined by use of Rose culture chambers (1). Dissociated primary spermatocytes were cultured at low cell-density in a collagen matrix at 22°C or 27°C and the percentages of cells which had progressed from various stages in prophase through meiosis to various advanced stages were measured. In a standard medium (Leibovitz-15 + 10% fetal bovine serum), more than 70% of the primary spermatocytes at stages beyond the pachytene stage could advance to round spermatids with flagella within a few days at 22°C. The percentages of cells that progressed from stages before the late zygotene stage were less, but at least 13 % of leptotene cells reached metaphase I within a week at 22°C. The percentage of cells that progressed was slightly lower at 27°C than at 22°C: 6.3 and 4.3 days were required for progress from leptotene to metaphase I at 22°C and 27C, respectively. Fetal bovine serum was not indispensable for progression through meiosis. Moreover, 0.5–5.0 μg/ml ovine follicle stimulating hormone (NIAMDD-o-FSH-13), 0.01–1.0 μg/ml 5α-dihydrotestosterone and 1.0 μg/ml testosterone propionate had no significant effect in increasing the percentage of cell progression at 22°C.     

The Behaviour of Dissociated Chick Embryo Brain Cells in Long-term Cultures in Presence and Absence of Brain Extracts

Dissociated nerve cells from 7 day old chick embryo cerebral hemispheres have been cultivated in Rose chambers. These cells have been maintained in a stable differentiated state for several months in minimal nutrient medium.
Brain extracts from chick embryos, and from new born rats, added to the nutrient medium during the first 2 weeks, stimulated the differentiation of neurones and glial cells. These well-differentiated nerve cells have also been maintained for very long periods in culture.
From these morphological observations, it has been concluded that dissociated nerve cells cultivated in Rose chambers provide good experimental models for electrophysiological investigations.     

Local expression of cytokeratins 8, 17 and 18 in the mesenchyme and smooth muscles in the early stages of human organogenesis

Expression of cytokeratins 7, 8, 17, 18 in human embryos and fetuses of 6.5-13 weeks was studied using light and electron immunocytochemistry and immunoelectroblotting with the monoclonal antibodies. Cytokeratins 8 and 18 were expressed in 6.5-8 week old embryos not only in epithelium but also in mesenchyme of allantois, urogenital sinus, Wolffian and Mullerian ducts, mesentery, urinary bladder and certain regions of colon, rectum and atrium cordis walls. Furthermore, starting from the 10th week smooth-muscle cells of ring layer in caudal part of rectum bound antibodies against cytokeratin 17 in addition to those against cytokeratins 8 and 18. Corresponding mesenchymal and smooth-muscle cells of adult individuals did not react with either of them. Cytokeratins were still synthesized when mesenchymal cells of embryonic intestine wall were cultivated in vitro. Intermediate filaments of these cells contain cytokeratins 8 and 18, as demonstrated by electron immunocytochemistry and immunoelectroblotting. Thus, the expression of cytokeratins is not restricted to adult and embryonic epithelial tissues but is also characteristic of mesenchyme and smooth muscle differentiation in human embryos and fetuses.     

Effects of co-culture and embryo number on the in vitro development of bovine embryos

It is generally accepted that culturing embryos in groups or with somatic cells improves both the yield and quality of the blastocysts obtained. The aims of this study were 1) to compare the yield and quality of the embryos obtained after culture in several number conditions and in several culture systems and 2) to assess the effect of co-culture started at various stages of embryo development. Under cell-free culture conditions (modified synthetic oviduct fluid [mSOF] supplemented with 10% fetal calf serum [FCS] 48 h post insemination, the rate of Day 10 blastocysts was lower when embryos were cultured in small groups (1 to 6 per drop) than in large groups (4 versus 23% ; P < 0.01). There was no group effect when embryos were co-cultured either with Buffalo rat liver (BRL) cells in TCM 199, or in a culture system allowing the progressive development of cumulus cells in mSOF, even if co-culture started at 66 or 114 h post insemination. However, embryos cultured singly had lower cell numbers than embryos cultured in large groups when co-culture started at 114 h post insemination. This suggests that 1) somatic cells improve the development of singly cultured bovine embryos up to the blastocyst stage after the 9-16 cell stage; 2) co-culture affects blastocyst cell number of singly cultured embryos by acting roughly between the 5-8 and the 9-16 cell stage; and 3) cooperation between embryos could replace the effect of co-culture either on the yield of blastocysts or on blastocyst cell number. Blastocysts appeared significantly earlier in co-culture with cumulus cells in mSOF than in co-culture with BRL cells in TCM 199 (detection of the blastocysts: 7.3 +/- 0.1 d post insemination with cumulus cells versus 8.1 +/- 0.1 d with BRL cells; P < 0.001) and had a significant higher number of cells (143 +/- 9 versus 85 +/- 11; P < 0.001). This system thus seems suitable for the culture of small numbers of embryos resulting from in vitro maturation and fertilization of oocytes from individual donor cows.     

Epithelial-Directed Mesenchyme Differentiation in vitro

To assess the requirement for specific or possibly non-specific epithelial instructions for mesenchymal cell differentiation, we designed studies to evaluate and compare homotypic with heterotypic tissue recombinations across vertebrate species. These studies further tested the hypothesis that determined dental papilla mesenchyme requires epithelial-derived instructions to differentiate into functional odontoblast cells using a serumless, chemically-defined medium. Theiler stage 25 C57BL/6 or Swiss Webster cap stage mandibular first molar tooth organs or trypsin-dissociated, homotypic epithelial-mesenchymal tissue recombinants resulted in the differentiation of odontoblasts within 3 days. Epithelial differentiation into functional ameloblasts was observed within 7 days. Trypsin-dissociated and isolated mesenchyme did not differentiate into odontoblasts under these experimental conditions. Heterotypic recombinants between quail Hamburger-Hamilton stages 22–26 mandibular epithelium and Theiler stage 25 dental papilla mesenchyme routinely resulted in odontoblast differentiation within 3 days in vitro. Odontoblast differentiation and the production of dentine extracellular matrix continued throughout the 10 days in organ culture. Ultrastructural observations of the interface between quail and mouse tissues indicated the reconstitution of the basal lamina as well as the maintenance of an intact basal lamina during 10 days in vitro. Quail epithelial cells did not differentiate into ameloblasts and no enamel extracellular matrix was observed. These results show that quail mandibular epithelium can provide the required developmental instructions for odontoblast differentiation in the absence of serum or other exogenous humoral factors in a chemically-defined medium. They also suggest the importance of reciprocal epithelial-mesenchymal interactions during epidermal organogenesis.     

Minimal nutrient requirements for culture of one-cell rabbit embryos

The minimal nutrient requirements of one-cell rabbit embryos for cleavage during in vitro culture were investigated. One-cell rabbit embryos were cultivated in a simple salt solution supplemented with the macromolecule polyvinylalcohol (PVA) either alone or with bovine serum albumin (BSA), amino acids, or one of a number of potential energy sources. At the end of 48 h culture, the embryos were stained with aceto-orcein HCl and the number of nucleated cells per embryo counted. One-cell embryos in medium with PVA but without an exogenous, fixed nitrogen source or energy substrate cleaved to a mean of 10.4 cells per embryo. Addition of the putative energy substrates--phosphoenolpyruvate, malate, acetate, and lactate--resulted in nonsignificant increases in cleavage rate. Glucose, pyruvate, a group of 20 amino acids from Ham's F-10 medium, and BSA gave a statistically significant doubling of the cleavage rate. These results indicate that the one-cell rabbit embryo, unlike the mouse embryo, has significant endogenous energy sources and that an exogenous, fixed nitrogen source is not essential for cleavage.     

Meiosis of Primary Spermatocytes and Early Spermiogenesis in the Resultant Spermatids in Newt, Cynops pyrrhogaster in vitro

Dissociated spermatogenic cells were cultivated within the collagen matrix at low cell density. The largest cell type in the culture was identified as the primary spermatocytes by their size and the morphological characteristics revealed by ultra-thin sections. Chromosome analysis showed that about 90% of the cells examined were either in first or second meiosis. Within the collagen matrix, the fates of 282 single primary spermatocytes at meiotic stage in diakinesis or metaphase were followed. In a few days, most of them gave rise to four spermatids, passing through first and second meiotic divisions. About 80% of the spermatids formed motile flagella. They grew about 20–60 μm a day. The final state of the differentiation attained in our culture conditions was the spermatids with localized spherical nuclei and motile flagella, about 500 μm in length after 1-month's culture. Ultra-thin sections of the spermatids show that the rings, neck-pieces, and acrosomes developed in the cells.     

A tissue culture analysis of the steps in limb chondrogenesis

Summary Tissue-culture methods can be used to test the developmental capacity of embryonic cells. In micro-mass cultures, derived from wing cells of stages 21 through 24 chick embryos, aggregates of cells form and then differentiate into cartilage nodules, as judged by the presence of an Alcian blue staining extracellular matrix. Wing cells derived from embryos as young as stage 17 can form aggregates. However, unless they are treated with db cyclic AMP and theophylline, it is not until stage 20 that these aggregates can produce cartilage in culture. In clonal cell culture, cartilage colonies are not produced by primary cell suspensions of limb cells until stage 25 when overt cartilage differentiation is occurring in vivo. It is possible to obtain clonable cartilage cells from limb cells from embryos between stages 20 and 24 if the cells are either treated with db cyclic AMP and theophylline or maintained in suspension culture for 12 to 48 hr. On the basis of these in vitro results a multiple step model for the conversion of limb mesenchyme into cartilage cells is proposed. The model involves the appearance of cells with a predisposition to form aggregates, development of the capacity to form cartilage in response to elevated levels of cyclic AMP, the appearance of receptors that translate changes in either cell shape or cell cycle parameters into elevated levels of cyclic AMP, aggregation, elevated levels of cyclic AMP, cartilage cell determination, and differentiation. This model can serve as the basis for further tests. Presented in the Opening Symposium on Nutritional Factors and Differentiation at the 28th Annual Meeting of the Tissue Culture Association, New Orleans, Louisiana, June 6–9, 1977. This work was supported by USPHS Training Grant HD00152 from the National Institute of Child Health and Human Development, while P.B.A. was a postdoctoral trainee, and by NIH Grant HD05505 to M.S.     

Increasing carbon dioxide from five percent to ten percent improves rabbit blastocyst development from cultured zygotes.

One-cell rabbit zygotes were cultured at 39 degrees C in basal synthetic medium II (BSM-II) with 5%, 10%, or 15% CO2 and humidified air to determine the effect of CO2 concentration on development in vitro. After 4 days in culture, 37% of the embryos grown in 10% or 15% CO2 had reached the hatching blastocyst stage, but only 10% of the embryos were hatching when cultured under 5% CO2 (P = 0.01). Over all blastocysts, cell numbers were 207, 246, and 205 for the 5%, 10%, and 15% CO2 treatments, respectively. In a second experiment to determine if there was a beneficial effect, particularly at the blastocyst stage, of a higher concentration of CO2, embryos were cultured 4 days in either 5% or 10% CO2 or for 2 days in 5% CO2 followed by 2 days in 10% CO2. The numbers of blastomeres per embryo and embryo diameter were greater (P < 0.05) in embryos cultured continuously in 10% CO2 or in 10% CO2 only during days 3 and 4 of culture than in embryos cultured continuously in 5% CO2. In a third experiment, one-cell rabbit zygotes were cultured with 5% or 10% CO2 in a defined, protein-free medium consisting of 1:1 RPMI 1640 and Dulbecco's modified Eagle's medium. The proportion of embryos hatching and cell counts were significantly greater (P < 0.01) when cultured in the presence of 10% CO2. These data indicate that a 10% CO2 atmosphere exerts a beneficial effect on the development of zygotes into expanding and hatching rabbit blastocysts in vitro.