In vitro culture of bovine IVM-IVF embryos: Cooperative interaction among embryos and the role of growth factors

The objective of this study was to determine whether there is a cooperative interaction among bovine embryos during in vitro culture. Furthermore, culture medium was supplemented with the growth factors, epidermal growth factor (EGF) and transforming growth factor-beta1 (TGF-beta1), to determine if these factors had a stimulatory effect on bovine embryo development similar to that seen in mouse development. In vitro matured - in vitro fertilized bovine embryos (2- to 8-cell) were cultured singly and in groups of five in 25 mul of medium (CR1 + amino acids + fatty acid-free bovine serum albumin) with or without EGF and TGF-beta1. Bovine embryos cultured in groups had a significantly higher rate of development to the blastocyst stage than embryos cultured singly. Neither EGF (10 ng/ml) nor TGF-beta1 (2 ng/ml) affected blastocyst development, hatching or the cell number of the embryos cultured in groups. Epidermal growth factor stimulated hatching of embryos cultured singly from the 8-cell stage, but did not significantly affect blastocyst development.     

Effect of various growth-promoting factors on preimplantation bovine embryo development in vitro

One-cell bovine embryos produced by in vitro oocyte maturation (IVM) and in vitro fertilization (IVF) were cultured in chemically defined medium (CDM) to which the following growth-promoting factors were added separately: transferrin (Tf, 10 ug/ml), epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), transforming growth factor-betal (TGF-beta1), insulin-like growth factor-one (IGF-I), insulin-like growth factor-two (IGF-II), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and nerve growth factor (NGF), all at 10 ng/ml. The embryos were cultured for a total of 10 days and were assessed. The culture medium was changed every 2 days. There were no beneficial effects of growth factors on embryo development to morula or hatched blastocyst stages of development. However, supplementation with EGF approached significance (P<0.08) when compared with that of CDM alone at the blastocyst stage of development. The results suggest that supplementation with growth factors does not improve bovine IVM-IVF embryo development when cultured under chemically-defined conditions.     

Platelet derived growth factor (PDGF) stimulates development of bovine embryos during the fourth cell cycle.

In vitro produced, 2-cell bovine embryos were cultured in serum-free medium supplemented with various combinations of growth factors to test the hypothesis that these polypeptide factors are able to signal preimplantation development. The developmental arrest that occurs during the 8-cell stage with typical culture methods might be relieved by a growth factor-dependent mechanism that would stimulate expression of the embryonic genome, thereby mimicking events that occur in vivo in the oviduct during the fourth cell cycle (8- to 16-cell stage). Subsequently, other growth factors might promote compaction and blastulation, processes which normally occur in the uterus. The effects of growth factors on early embryos were evaluated using phase contrast microscopy to monitor progression to the 8-cell stage, completion and duration of the fourth cell cycle, and blastocyst formation. Platelet derived growth factor (PDGF) promoted development beyond the 16-cell stage in 39.1% of the 2-cell embryos examined in all experiments. The duration of the fourth cell cycle among these embryos was approximately 26 hours. During development after the 16-cell stage, PDGF reduced the proportion of embryos bastulating from 12.7% to 5.8%; in contrast, transforming growth factor alpha (TGF alpha), acting during the same developmental time period, increased the proportion of embryos blastulating from 8.6% to 40.6%. These results, using serum-free medium, indicated that PDGF signalled completion of the fourth cell cycle. TGF alpha, and perhaps basic fibroblast growth factor (bFGF), promoted blastulation of 16-cell embryos during subsequent culture.     

Secretion of stem cell factor and granulocyte-macrophage colony-stimulating factor by mouse embryos in culture: influence of group culture

Previous studies showed that the addition of a growth factor to the culture medium could modulate embryo development. The possible secretion of different factors to the culture medium by the embryo itself, however, has been poorly evaluated. The present study was designed to investigate: (1) the influence of single or group culture on the development of 2-cell mouse embryos (strain CD-1) to the blastocyst stage; (2) the release of granulocyte-macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF) into the culture medium by the embryo; and (3) the levels of GM-CSF and SCF in the culture medium from both single and group embryos. Two-cell CD-1 mouse embryos were cultured for 96 h singly or in groups of five embryos per drop. GM-CSF and SCF were assayed by ELISA in the complete culture medium. It was found that embryos cultured in groups gave a higher percentage of total blastocyst formation and hatched blastocyst when compared with single embryo culture. The mouse embryos secreted GM-CSF and SCF to the culture medium. The concentration of these cytokines is significantly higher in the group cultures than the level found in single cultures. In conclusion, mouse embryos in culture secrete GM-CSF and SCF to the culture medium and the concentration of these cytokines increases during communal culture. These factors may be operating in both autocrine and paracrine pathways to modulate embryo development during in vitro culture.     

Buffalo rat liver cells produce factors that support preimplantation development of mouse embryos cultured in vitro

To examine the effects of buffalo rat liver (BRL) cells on the preimplantation development of mouse embryos in vitro, we first cultured two-cell mouse embryos alone in serum-free Dulbecco modified Eagle medium. As expected, the embryos did not develop to subsequent stages. However, when cocultured with BRL cells, the embryos developed to the blastocyst stage efficiently. Direct contact of embryos with BRL cells was not necessary for development: the medium conditioned by BRL cells contained soluble factors that supported the preimplantation development of mouse embryos. Embryos cultured with BRL-conditioned medium that was replaced at various intervals had a further increased rate of development to the blastocyst stage. This finding indicated that the activities of the factors were maintained only briefly. Seven proteins between 35 and 44 kDa that were detected in the medium were highly beneficial to the development of the embryos. Follistatin-related protein and pigment epithelium-derived factor are believed to be the factors supporting embryo development. The other five proteins also may improve the environment for the development of mouse embryos cultured in vitro.     

Growth factor interactions between mouse mammary cell lines cocultured in collagen gels

Summary Three related mouse mammary cell lines were cultured in collagen gels and assayed for growth factor responsiveness and interaction via soluble factors. The CL-S1 cell line is nontumorigenic and grows poorly in collagen gel culture. The +SA and −SA cell lines exhibit different degrees of malignant behavior in vivo and have different growth properties in vitro. In collagen gel culture, +SA growth was stimulated by serum but not by epidermal growth factor (EGF), whereas both serum and EGF were required for optimal growth of −SA cells of early passage number as well as CL-S1 cells. −SA cells of later passage repeatedly exhibited a change so as to no longer require serum while retaining EGF responsiveness. [¹²⁵I]EGF binding analyses indicated that CL-S1 cells bound EGF with less affinity than did −SA cells whereas +SA cells bound almost to ligand. When cell lines were maintained in separate collagen gels but shared the same culture medium, growth of +SA or −SA cells was slightly enhanced in the presence of CL-S1 cells and −SA cell growth was enhanced by the presence of +SA cells. Using the normal rat kidney fibroblast line NRK (clone 49F) as an indicator, serum-containing conditioned media from each cell line and from each pair of cell lines cultured in collagen gels were tested for transforming growth factor (TGF) activity. Both the −SA and CL-S1 lines tested positive for TGF-α production and possibly released a TGF-β activity. These results suggest mechanisms by which cell populations in and around tumors can modify one another’s growth characteristics. The work was supported by a grant from the American Institute for Cancer Research, by American Cancer Society Institutional grant IN-119, by funds from the Poncin Trust (Seattle-First National Bank), and by grants CA-39611 and CA46885 from the National Institutes of Health, Bethesda, MD.     

Embryonal carcinoma cell growth and differentiation. Production of and response to molecules with transforming growth factor activity

Transforming growth factors are known to induce anchorage-independent growth of non-transformed cells, and are released by a variety of cells, including MSV-transformed cells. This study demonstrates that the differentiated cells derived from F9 and PC-13 embryonal carcinoma cells, but not the parental cells themselves, respond by increased growth to several factors released by MSV-transformed cells, including partially purified sarcoma growth factor. The chemical properties of the growth-promoting activity are shown to match the chemical properties of the transforming growth factors released by MSV-transformed cells. Furthermore, F9 and PC-13 embryonal carcinoma cells, which do not respond to factors released by MSV-transformed cells, are shown to release factors with transforming growth factor activity. Based on the close relationship between mouse embryonal carcinoma cells and cells of early mouse embryos, it is suggested that molecules with transforming growth factor activity may play a role during the early stages of mammalian development.     

Hypothermia: teratogenic and protective effects on the development of mouse embryos in vitro

Hypothermia often occurs in association with clinical conditions involving severe hypoglycemia, but its effect on embryonic development has not been well evaluated. Thus, the whole embryo culture method was used to expose day 9 (neurulating) and day 10 (early limb bud stage) mouse embryos to physiologic levels of hypothermia (35 degrees C and 32 degrees C) for 4 and 24 hr. Embryos were evaluated after 24 hours for growth and malformations and compared with controls grown at 37 degrees C. Lactate production was measured in embryos cultured for 4 hr at 32 degrees C and compared with those cultured at 37 degrees C. A 4-hr exposure to hypothermia produced little effect morphologically but reduced the rate of lactate production at both embryonic stages. A 24-hr exposure to hypothermia at 35 degrees C or 32 degrees C produced growth retardation and dysmorphogenesis in embryos undergoing neurulation. Early limb bud stage embryos were less sensitive to this treatment, with growth retardation produced only at the lower temperature. Since hypothermia is commonly associated with severe hypoglycemia in cases of diabetic insulin overdose, day 9 (neurulating) mouse embryos were exposed concurrently to short periods of hypothermia and hypoglycemia and compared with embryos cultured in hypoglycemic medium at normal temperature. The results demonstrate that hypothermia partially protects embryos against the dysmorphogenic effects of hypoglycemia. A balance of metabolic rate and available substrate is discussed as a possible mechanism for this protective effect.     

Effects of fibroblast growth factor 2 and insulin-like growth factor II on the development of parthenogenetic mouse embryos in vitro

Most parthenogenetic embryos (PEs) in mammals die shortly after implantation, and this failure to develop is associated with genomic imprinting. We have examined the influence of human recombinant basic fibroblast growth factor 2 (FGF-2) and human recombinant insulin-like growth factor II (ICF-II) on the development of (CBA x C57BL/6)F1 parthenogenetic mouse embryos. Embryos were treated in vitro at the morula stage with different doses of FGF-2 and, after their development to blastocysts, transferred to pseudopregnant recipients. The optimal doses of FGF-2 did not affect the number of forming and implanting blastocysts, but increased, from 20 to 42%, the number of embryos developing to somite stages. PEs (18-21 somites) treated with an optimal dose of FGF-2 were explanted for further development in culture by treatment with the second growth factor, IGF-II. Eighty-three percent of those embryos cultured with IGF-II (2.5 microg/ml) developed to 35 or more somites, as compared with 36% of embryos cultured without any growth factors (P < 0.01). Also, a significantly higher proportion of PEs developed to 40-50 somites in this case. These results show that the in vitro treatment of PEs with FGF-2 at the morula stage increases the number of somite embryos, and the second treatment of somite PEs with IGF-II in culture medium prolongs their development significantly.     

Influences of retrieval stages and glutathione addition on post-thaw viability of quick frozen mouse morula during in vitro culture

Effects of the embryo retrieval stages and addition of glutathione (GSH) on post-thaw development of mouse morula were evaluated in 2 consecutive experiments. In the first experiment, 1-, 2-, 3- to 4- and 5- to 8-cell stage embryos were collected and cultured to the morula stage in Whitten's medium containing 0.1 mM ethylenediaminetetraacetic acid (EDTA). The development rate of 1-cell embryos to the morula stage was lower than that of the other stages (P<0.01). The post-thaw development rate of the morulae obtained from in vitro culture of 1-, 2-, 3- to 4-, and 5- to 8-cell embryos and from in vivo embryos (control) to the blastocyst stage was 55.5, 84.9, 87.4, 90.1 and 90.8%, respectively. The post-thaw development rate of morula obtained from in vitro produced 1-cell embryos was significantly lower than from the other stages or from the in vivo counterparts (P<0.0001). In Experiment 2, the impact of GSH supplementation of the culture medium in the presence or absence of EDTA was evaluated for embryo development to the morula stage and post-thaw survival, using in the 2 x 2 factorial design. Although EDTA supplementation increased development rates to the morulae (P<0.01) stage, GSH did not have an influence on morula development. However, the presence of either GSH or EDTA in the culture medium supported development to the blastocyst stage (P<0.01) of in vitro produced morulae. These data demonstrate that 1-cell embryos from a blocking-strain mouse cultured in vitro to the morula stage have a lower development rate following freezing and thawing than embryos collected at the 2-cell or later stages. Addition of EDTA or GSH, individually or in combination, to the culture medium may improve the development rate of morula to blastocyst stage following cryopreservation.     

Transforming growth factor beta and basic fibroblast growth factor synergistically promote early bovine embryo development during the fourth cell cycle.

Developmentally competent bovine blastocysts were produced by adding transforming growth factor beta (TGF beta) and basic fibroblast growth factor (bFGF) to serum-free cultures of in vitro produced, 2-cell bovine embryos. The effects of TGF beta were evaluated because this growth factor signals synthesis and secretion of the extracellular matrix component fibronectin and its receptor. Previous investigations have demonstrated that fibronectin promotes early bovine embryo development in vitro. The effects of TGF beta can be potentiated by bFGF; bFGF itself is an effector of protein synthesis and a potent mitogen. A positive interaction between the 2 growth factors resulted in 38.8% of fertilized oocytes maturing beyond the 16-cell stage; of these, 24.6% formed blastocysts. Transfer of early blastocysts produced using serum-free medium supplemented with growth factors resulted in pregnancy in 3 of 9 recipients. These results support the hypothesis that TGF beta and bFGF act synergistically to promote development of bovine embryos beyond the "8-cell block" observed in vitro.     

Embryo development from discrete cell aggregates inIpomoea Batatas (L.) Lam. in response to structural polarity

Summary High numbers of embryos are difficult to obtain in liquid cultures of sweet potato (Ipomoea batatas (L.) Lam.) because discrete cell aggregates, produced through calli fragmentation, do not support embryo growth. In an effort to demonstrate that embryo development is possible from discrete cell aggregates, we compared embryo formation from cell aggregates 250–355 μm in diameter cultured either in suspension in liquid medium, on agar solidified medium, or immobilized on alginate beads floated in liquid medium. Embryos were initiated but remained arrested in their globular stage on cell aggregates cultured in suspension. Embryos developed to the torpedo stage from cell aggregates cultured on solidified medium and from cell aggregates anchored on alginate beads. Thus, embryos continued to develop beyond the globular stage when a structural polarity, which led probably to the establishment of a physiological polarity, was created. The production of sweet potato embryos in liquid culture can be improved by using alginate beads or culture conditions and protocols leading to the release during calli fragmentation of polarized individual cell aggregates. This work was supported in part by a IFAS/Gas Research Institute cooperative grant. Florida Agriculture Experiment Station Journal Series 9297     

Protein-free medium for C-1300 mouse neuroblastoma cells

Summary An optimized medium, designated MCDB 411, has been developed for mouse neuroblastoma cells. At cell densities of 1×10⁴ cells/cm² or greater, several different clones of C1300 mouse neuroblastoma cells can be cultured serially in Medium MCDB 411 with no serum or other undefined supplementation and with a doubling time of about 24 h. At clonal densities it is necessary to supplement the medium with 1.0 μg/ml insulin. Alternately, good clonal growth can be obtained without direct supplementation by coating the culture dishes with insulin and rinsing off all that is not tightly bound. Primary cultures of cells from serially transplanted C1300 tumors that have never been cultured previously in vitro can be established directly in unsupplemented Medium MCBD 411 with rapid initiation of multiplication and no apparent crisis or selection for minority cell types. The availability of a synthetic medium that supports growth of neuroblastoma cells without supplementation should facilitate the use of these cells as model systems for the study of neuronal function and differentiation. This work was supported by Grant CA-15305 from the National Cancer Institute.     

Leukemia Inhibitory Factor (LIF) Improves and Prolongs the Development of Parthenogenetic Mouse Embryos

We studied the effect of the growth factor LIF on the development of parthenogenetic mouse embryos (CBA × C57BL/6)F1. LIF was added to the culture medium at 10, 50, 100, and 250 ng/ml at the morula stage and parthenogenetic embryos were cultured in vitro until the late blastocyst stage and then transplanted in the uterus of pseudopregnant females, which were then sacrificed on day 12 of pregnancy. All the LIF doses used improved the development of parthenogenetic mouse embryos at the preimplantation stages and increased the amount of blastocysts by 15%, on average, as compared to the control. LIF at 50 and 100 ng/ml increased approximately twice the number of embryos that reached the somite stages. Some of them reached the stage of 32–45 somites and had fore and hind limb buds. No such embryos were found in the control. Well formed placenta was observed in 6% of the embryos treated with LIF and the most pronounced effect was recorded at 100 ng/ml. The data we obtained suggest that exogenous LIF can improve pre- and postimplantation development of parthenogenetic mouse embryos due, possibly, to increased survival rate of embryonic stem cells derived from the inner cell mass of blastocysts. LIF improves not only the development of the parthenogenetic embryoper se, but also the formation of its extraembryonic envelopes, which leads to the development of a larger placenta in LIF-treated parthenogenetic embryos, as compared to the control.     

Nuclear-cytoplasmic "tug of war" during cloning: effects of somatic cell nuclei on culture medium preferences of preimplantation cloned mouse embryos

Cloning by somatic cell nuclear transfer is critically dependent upon early events that occur immediately after nuclear transfer, and possibly additional events that occur in the cleaving embryo. Embryo culture conditions have not been optimized for cloned embryos, and the effects of culture conditions on these early events and the successful initiation of clonal development have not been examined. To evaluate the possible effect of culture conditions on early cloned embryo development, we have compared a number of different culture media, either singly or in sequential combinations, for their ability to support preimplantation development of clones produced using cumulus cell nuclei. We find that glucose is beneficial during the 1-cell stage when CZB medium is employed. We also find that potassium simplex optimized medium (KSOM), which is optimized to support efficient early cleavage divisions in mouse embryos, does not support development during the 1-cell or 2-cell stages in the cloned embryos as well as other media. Glucose-supplemented CZB medium (CZB-G) supports initial development to the 2-cell stage very well, but does not support later cleavage stages as well as Whittten medium or KSOM. Culturing cloned embryos either entirely in Whitten medium or initially in Whittens medium and then changing to KSOM at the late 4-cell/early 8-cell stage produces consistent production of blastocysts at a greater frequency than using CZB-G medium alone. The combination of Whitten medium followed by KSOM resulted in an increased number of cells per blastocyst. Because normal embryos do not require glucose during the early cleavage stages and develop efficiently in all of the media employed, these results reveal unusual culture medium requirements that are indicative of altered physiology and metabolism in the cloned embryos. The relevance of this to understanding the kinetics and mechanisms of nuclear reprogramming and to the eventual improvement of the overall success in cloning is discussed.     

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O₂ / 5% CO₂ in a rolling bottle culture apparatus at 37 °​C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.     

An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro

One-cell CF-1 x B6SJLF1/J embryos, which usually exhibit a 2-cell block to development in vitro, have been cultured to the blastocyst stage using CZB medium and a glucose washing procedure. CZB medium is a further modification of modified BMOC-2 containing an increased lactate/pyruvate ratio of 116, 1 mM-glutamine and 0.1 mM-EDTA but lacking glucose. Continuous culture of one-cell embryos in CZB medium allowed 83% of embryos to develop beyond the 2-cell stage of which 63% were morulae at 72 h of culture, but blastocysts did not develop. However, washing embryos into CZB medium containing glucose after 48 h of culture (3-4-cell stage) was sufficient to allow development to proceed, with 48% of embryos reaching the blastocyst stage by 96 h of culture. Exposure of embryos to glucose was only necessary from the 3-4-cell stage through the early morula stage since washing back into medium CZB without glucose at 72 h of culture still promoted the development of 50% of embryos to the blastocyst stage. The presence of glucose in this medium for the first 48 h of culture (1-cell to 4-cell stage) was detrimental to embryo development. Glutamine, however, exerted a beneficial effect on embryo development from the 1-cell to the 4-cell stage although its presence was not required for development to proceed during the final 48 h of culture. Blastocysts which developed under optimum conditions contained an average of 33.7 total cells. The in-vitro development of 1-cell embryos beyond the 2-cell stage in response to the removal of glucose and the addition of glutamine to the culture medium suggests that glucose may block some essential metabolic process, and that glutamine may be a preferred energy substrate during early development for these mouse embryos.     

Development of hamster two-cell embryos in the isolated mouse oviduct in organ culture system

Hamster early two-cell embryos developed to the expanded blastocyst stage within the isolated mouse ampulla maintained in organ culture system. Mouse ampullae isolated at different times after treating the mice with human chorionic gonadotropin (hCG) (0–72 h) or pregnant mare's serum gonadotropin (PMSG) (30–32 h) were flushed with culture medium, and hamster early two-cell embryos were introduced into these ampullae. Mouse ampullae isolated at 14–32 h after hCG injection were more favorable for the development of the embryos than those isolated at 70–72 h. When mouse ampullae were isolated 30–32 h after hCG or PMSG treatment, 39% of the cultured eggs developed, some of them to the expanded blastocyst stage after additional culture for 65–70 h. These results indicate that unknown oviductal factors stimulate the development of hamster early two-cell embryos, and these factors are under the control of hCG or PMSG. In addition, these factors are common to the mouse and hamster.