Protein secretion by the mouse trophoblast during attachment and outgrowth in vitro

Protein secretion from mouse blastocysts undergoing attachment and trophoblast outgrowth in vitro was assessed. When Day 5 blastocysts were cultured in serum-containing medium, secretion of several 'attachment-associated' proteins (PAS) was initiated within 24 h, coincident with attachment and outgrowth. Those proteins characteristic of the pre-attachment blastocyst disappeared or made-up only a small portion of the secretions once attachment began. The major secreted protein from attached embryos, PA1, is a 35,000-45,000 Mr acidic glycoprotein with multiple isoelectric forms. PA2, a group of basic 40,000 Mr proteins and PA3 a group of 72,000 Mr proteins were also produced during outgrowth. PAS were secreted during outgrowth on fibronectin-coated plastic in serum-free medium, but not by blastocysts held in a non-attachment state during culture in serum-free medium on uncoated plastic. In pre-attachment blastocysts, secreted proteins were produced by trophoblast vesicles, but not by isolated inner cell masses. Both trophoblast vesicles growing out in vitro and surgically isolated trophoblast from spreading blastocysts had secreted protein patterns qualitatively similar to those of intact blastocyst outgrowths. The results indicate that development of trophoblast protein secretion continues through the period of outgrowth and giant cell transformation. These changes are apparently dependent on attachment of the blastocyst to a suitable substrate, but not dependent on any other serum influence.     

In vitro culture and interferon-tau secretion by ovine blastocysts

Embryos were collected surgically from superovulated ewes on days 7, 8, 9 and 10 (oestrus=day 0) to evaluate the long-term culture and interferon-tau (IFN-τ) secretion of ovine blastocysts. Embryos were cultured in 2 ml Dulbecco’s modification of Eagle’s medium (DMEM) supplemented with 15 mg/ml BSA in 5% CO₂ in air or DMEM without BSA in 5% CO₂, 7% O₂, and 88% N₂ at 39 °C, examined daily for morphological features and diameter and each day placed into fresh culture medium to enable daily measurement of IFN-τ secretion. Nine day-7 and two day-9 embryos were cultured in DMEM with BSA and nine continued to develop. The day-7 embryos reached a mean maximum diameter of 370.0±50.25 μm after 4 days in culture. Nineteen day-7, 12 day-8 and five day-10 embryos were cultured in DMEM without BSA but only six of the day-7 and one day-8 embryos survived for at least 7 days with the former reaching a mean maximum diameter on day 7 of 357±43.75 μm whereas all five day-10 embryos survived for at least 7 days reaching a mean maximum diameter on day 6 of 1038±155.8 μm. An anti-viral assay and a ELISA for IFN-τ were developed. There was a considerable variation in the time of onset and amount of IFN-τ secreted that did not seem to be related to embryo morphology. Of 28 day-7 embryos cultured, 60.7% were secreting IFN-τ after 1 day of culture whereas 87.5% of day-8 embryos were secreting IFN-τ after 1 day in culture. The mean concentration of IFN-τ secreted by day-8 embryos after 1 day in culture (10.99±2.55 ng/ml) was not significantly different to day-7 embryos after 2 days in culture (8.8±1.75 ng/ml).     

Effects of conditioned media on porcine embryos at different stages of development

The objective of our study was to determine the effect of conditioning media with homologous porcine uterine cells on the developmental rate of porcine embryos. Cell monolayers were prepared by selective dissection and digestion of sections from the uterus of prepuberal gilts that were primed with PMSG and hCG. Conditioned media were used with 2 type of embryos: 4-cell stage (Experiment 1) or blastocyst stage (Experiment 2). In Experiment 1, embryos were collected surgically by flushing the oviducts, 36 to 48 h following the first of 2 inseminations. Embryos were cultured in Whitten's medium containing 1.5% BSA as a protein source until they attained the 4-cell stage. Embryos at the 4-cell stage were cultured randomly in either Whitten's medium with 1.5% BSA or Whitten's medium with 1.5% BSA that was previously conditioned for 24 h with an endometrial epithelial cell monolayer. Embryos were cultured in 50-microl drops covered with oil in a 38.5 degrees C, 5% CO(2) in air incubator. There was no advantage to using the conditioned media with the 4-cell stage embryos. The embryos were less developed than those cultured in nonconditioned Whitten's medium (P <0.001). In Experiment 2, embryos were cultured at the blastocyst stage. They were recovered the same way as in Experiment 1 and then cultured in Whitten's medium containing 1.5% BSA until they reached the blastocyst stage. At the blastocyst stage (Day 6), embryos were randomly assigned to 1 of the 6 following treatments: Whitten's with 1.5% BSA or Whitten's plus 1.5% BSA that was previously conditioned with endometrial epithelial cell monolayer, TCM-199 containing 0.4% BSA or TCM-199 plus 0.4% BSA that was previously conditioned with endometrial epithelial cell monolayer, finally, TCM-199 containing 10% serum or TCM-199 plus 10% serum that was previously conditioned with endometrial epithelial cell monolayer. Results show that initiation of hatching was significantly enhanced by conditioning the Whitten's media.     

Genetic and environmental determinants of interferon-tau secretion by in vivo- and in vitro-derived bovine blastocysts

Several experiments were conducted to assess the effects of genotype and various culture media on interferon-tau secretion by in vitro-derived bovine blastocysts and to compare these values with interferon released by blastocysts flushed from superovulated cows. In experiment 1, oocytes were inseminated with semen from three different bulls. While paternal genotype had no effect on cleavage rate, the size or hatching ability of blastocysts, it was a significant determinant of the embryo's ability to develop to the blastocyst stage and of subsequent interferon-tau secretion. In the second experiment, embryos were cultured in synthetic oviductal fluid containing either polyvinyl alcohol, bovine serum albumin or fetal bovine serum. While there was no effect of supplement on the percentage of embryos developing to the blastocyst stage, blastocysts which formed in medium with polyvinyl alcohol had significantly fewer cells, were older at blastocyst formation and produced significantly more interferon-tau. In the third experiment, embryos were cultured to the blastocyst stage in either TCM199 alone or in co-culture with buffalo rat liver, bovine oviductal or bovine uterine epithelial cells. Culture with oviductal or buffalo rat liver cells increased blastocyst cell number, although secretion of interferon-tau was not affected. In the final experiment, bovine blastocysts were flushed from superovulated cows on Day 7 following insemination. Overall, secretion of interferon-tau by in vivo-produced blastocysts did not differ from that of age-matched blastocysts produced in vitro.     

Lipid content in pig blastocysts cultured in the presence or absence of protein and vitamin E or phenazine ethosulfate

In the present study, total lipid content and content of triglycerides, phospholipids and cholesterol were determined in pig blastocysts cultured in medium without protein, supplemented with bovine serum albumin (BSA), with fetal calf serum (FCS), vitamin E or phenazine ethosulfate (PES). In comparison to blastocysts cultured in NCSU-23 with BSA, we observed a decrease of the total lipid content in PES-treated embryos. Triglyceride content in FCS-, vitamin E- and PES-treated embryos as well as in blastocysts cultured without protein was 81.9%, 70.2%, 57.2% and 74.8% of that found in the blastocysts cultured in NCSU-23 with BSA, respectively. Nevertheless the content of phospholipids remained unchanged. This decrease of triglyceride content in the porcine blastocyst after in vitro culture may be explained by altered lipid metabolism in embryos.     

Development and subsequent cryotolerance of domestic cat embryos cultured in serum-free and serum-containing media

The objectives of this study were to examine the effects of the presence or absence of serum during the in vitro culturing period of domestic cat embryos on their developmental potential into blastocysts as well as their tolerance to cryopreservation using a slow-freezing method. In vitro-fertilized cat oocytes were incubated in a modified synthetic oviduct fluid (mSOF) containing 4 mg/mL bovine serum albumin (BSA) throughout culturing (BSA group) or in mSOF containing 4 mg/mL BSA for the first 3 days followed by mSOF containing 5% fetal bovine serum (FBS group). The developmental potential of the embryos to the blastocyst and expanded blastocyst stages was evaluated 7 days after in vitro fertilization. The blastocysts were frozen-thawed by the slow-freezing method and cultured for 3 days to examine their viability in vitro. There were no differences in the formation rates of blastocysts or expanded blastocysts, or number of cells in the embryos between the two groups. After cryopreservation, the hatching rates of the expanded blastocysts in the BSA group were significantly higher (P < 0.05) than those of the FBS group. The postthaw viability of blastocysts was lower than that of expanded blastocysts irrespective of culture medium. These results indicate that the developmental potential of cat embryos cultured in serum-free medium is comparable to those cultured in serum-containing medium. Furthermore, expanded blastocysts produced without serum exhibit better postthaw viability than those produced with serum.     

Effects of glucose and protein sources on bovine embryo development in vitro

Oviductal factors may be obtained by ultrafiltration of conditioned medium, added to a simple media and used in bovine embryo culture. In this study, we aimed to analyze the development of bovine embryos produced with oviductal factors compared to those cultured in the presence of BSA or serum, the effects of glucose in presence of these protein supplements, and the ability of oviductal factors to support embryo development during the entire culture period. In vitro produced bovine zygotes from slaughterhouse ovaries were cultured in modified-synthetic oviduct fluid (mSOF) alone or supplemented with (1) oviductal factors, (2) BSA and (3) FCS. Oviductal factors showed embryotrophic activity, although with blastocyst rates lower than those in BSA and FCS. Glucose (1.5 mM) added at Day 2 of culture did not affect development in the presence of oviductal factors. The number of cells in expanded blastocysts was unaffected by the presence of glucose or any of the protein supplements used. Both BSA and FCS, respectively, improved blastocyst rates of Day 6 embryos produced with oviductal factors. The effect of oviductal factors was masked by the presence of BSA during the entire culture. FCS promoted an earlier appearance of blastocysts. It is concluded that the effect of glucose on in vitro embryo development depends upon the source of protein. Oviductal factors are not an appropriate supplement for embryos beyond Day 6 of culture in SOF, although blastocyst rates of such embryos may be increased by culturing them in the presence of FCS or BSA.     

Initiation of placental lactogen-I production by blastocysts growing on a two-dimensional surface and in hanging drops

The mouse blastocyst undergoes a program of protein secretion during the perimplantation period including the initial production of placental lactogen-I (mPL-I) on day 6 of pregnancy. Although blastocysts collected on day 5 also produce mPL-I when cultured to form outgrowths on plastic dishes, it was not known if embryos have an intrinsic ability to produce mPL-I during culture in vitro, or if a specific uterine influence is necessary. Earlier experiments also suggested that attachment of the trophoblast to a stable surface may be a prerequisite for synthesis of mPL-I. Both questions were addressed by examining mPL-I production by day 3 and day 4 embryos cultured either on a two-dimensional tissue culture dish surface or in hanging drops. The presence of intracellular mPL-I was assayed by immunohistochemistry, while the secreted hormone was detected by its known position in two-dimensional electrophoresis gels. These experiments demonstrated that these earlier stage embryos do have an intrinsic program for mPL-I production which proceeds in vitro under various culture conditions. Synthesis of mPL-I occurred in embryos suspended in hanging drops as well as in those spreading on a solid two-dimensional surface, thus showing that adhesion to a surface is not required for production of this hormone. Although some mPL-I synthesis was seen in embryos cultured in medium containing BSA as the sole macromolecule, the inclusion of fibronectin either in dishes, where it supports attachment, or in the hanging drop system stimulated mPL-I secretion. Serum supplementation in both culture systems further increased growth and differentiation of the embryo, as well as mPL-I secretion, compared to fibronectin supplementation.     

Factors influencing the in vitro hatching of mouse blastocysts

The influence of bovine serum albumin (BSA) concentration on embryo hatching and the number of embryos cultured per drop of culture medium was examined in F1 (C57BL/6J × DBA/2J), C3HeB/FeJ strain and Line E mice. Embryos collected from F1 and Line E mice exhibited uniform hatching rates at BSA concentrations between 1 and 10 mg/ml, and embryo numbers ranging from 1 to 10 per 3 μ1 of culture medium. The hatching of C3HeB/FeJ blastocysts was greater at the higher concentrations of BSA and higher embryo densities. When the C3HeB/FeJ embryos were grown at high densities until morula and then cultured singly in fresh media they hatched at a low rate. However, when allowed to develop until the blastocyst stage before replotting, the embryos hatched at a high rate. C3HeB/FeJ embryos cultured singly until morula and then placed in groups of 10 hatched at a high rate. Single C3HeB/FeJ embryos, cultured in medium conditioned by the prior presence of embryos at high densities, hatched at a slightly higher frequency than those cultured in fresh medium. There was no tendency of embryos developing from the two-cell to the eight-cell stages to hatch when cultured in the presence of high densities of hatching blastocysts.     

Differential patterns of blastulation in bovine morulae cultured in synthetic oviduct fluid medium containing FCS or BSA

This study examined the effects of fetal calf serum (FCS) supplementation of culture medium on blastulation and hatching of bovine morulae cultured in vitro. The presumptive zygotes derived from in vitro maturation and fertilization (IVM/IVF) were cultured in the modified synthetic oviduct fluid medium containing 3 mg/ml BSA (mSOF-BSA). At 120 h post insemination, morulae were randomly assigned to culture with mSOF-BSA (control) or mSOF containing 5% FCS (mSOF-FCS) instead of BSA. The replacement of BSA with FCS in mSOF significantly increased the percentage of blastocyst formation from Day 6 to Day 10 (Day 0 = the day of in vitro insemination) and the hatching rate of embryos on Days 8 and 9. The total number of cells in morulae and blastocysts on Day 6, in blastocysts on Day 7, and in blastocysts and hatched blastocysts on Day 8 were similar among the treatments. However, the replacement of BSA with FCS in mSOF significantly increased the total number of cells in hatched blastocysts on Day 10. Although the time of blastulation of embryos was significantly accelerated by the replacement of BSA with FCS in mSOF, the total number of cells in embryos at blastulation was lowered. The total number of cells in embryos at blastulation showed a time-dependent decrease when the embryos were cultured in mSOF-BSA. In contrast, the total number of cells in embryos that were cultured in mSOF-FCS depended little on the time after in vitro insemination. The results indicate that FCS supplementation of culture medium increased the percentage of embryos developing to the blastocyst stage without an increase in the total number of cells. However, an acceleration in the hatching rate and an increase in the total number of cells in hatched blastocysts were observed, compared with that in BSA-supplemented medium. It is suggested that FCS in the culture medium initiates earlier blastulation with fewer total numbers of cells in the morulae than BSA during in vitro culture of bovine embryos.     

Development and subsequent cryotolerance of domestic cat embryos cultured in serum-free and serum-containing media

The objectives of this study were to examine the effects of the presence or absence of serum during the in vitro culturing period of domestic cat embryos on their developmental potential into blastocysts as well as their tolerance to cryopreservation using a slow-freezing method. In vitro-fertilized cat oocytes were incubated in a modified synthetic oviduct fluid (mSOF) containing 4 mg/mL bovine serum albumin (BSA) throughout culturing (BSA group) or in mSOF containing 4 mg/mL BSA for the first 3 days followed by mSOF containing 5% fetal bovine serum (FBS group). The developmental potential of the embryos to the blastocyst and expanded blastocyst stages was evaluated 7 days after in vitro fertilization. The blastocysts were frozen-thawed by the slow-freezing method and cultured for 3 days to examine their viability in vitro. There were no differences in the formation rates of blastocysts or expanded blastocysts, or number of cells in the embryos between the two groups. After cryopreservation, the hatching rates of the expanded blastocysts in the BSA group were significantly higher (P < 0.05) than those of the FBS group. The postthaw viability of blastocysts was lower than that of expanded blastocysts irrespective of culture medium. These results indicate that the developmental potential of cat embryos cultured in serum-free medium is comparable to those cultured in serum-containing medium. Furthermore, expanded blastocysts produced without serum exhibit better postthaw viability than those produced with serum.     

Protein-free culture medium containing polyvinylalcohol, vitamins, and amino acids supports development of eight-cell hamster embryos to hatching blastocysts

In vitro development of eight-cell hamster embryos to hatching blastocysts requires the presence of amino acids and a group of water-soluble vitamins in the culture medium. The present studies investigated the effect of type of macromolecule on blastocyst hatching and on the requirement for vitamins. Embryos were cultured for 3 days in the presence of the synthetic macromolecule polyvinylalcohol (PVA) and of different types of bovine serum albumin (BSA), both with and without vitamins. The results showed th at eight-cell embryos develop to hatching blastocysts in the presence of vitamins and amino acids with PVA as the only macromolecule in the medium. The presence of certain types of BSA reduced but did not eliminate the need for vitamins. Glutamine alone was as efficient as a complete amino acid supplement in supporting blastocyst hatching. These results demonstrate for the first time that eight-cell hamster embryos can be cultured to hatching blastocysts in a chemically defined medium.     

Embryo survival, uterine fluids and tubal SEM in progesterone-asynchronized rabbits

Survival of embryos exposed to several concentrations of uterine proteins and changes in tubal morphology in rabbits given low preovulatory doses of progesterone (P4) that had previously not affected ovulation or fertilization, but caused severe embryo mortality, were studied. In experiment 1, 332 morulae were cultured for 24 h in a control medium containing < 0.5 to > 3.0 mg x mL(-1) of Day 3 uterine fluid proteins. There was no difference in blastocyst development nor implantation to Day 12 following transfer of the blastocysts to recipients, except fewer implants developed in the BSA control. In experiment 2 the oviducts and uteri of control and P4-treated does were examined by SEM for 8 days following ovulation. Secretory cells in the oviducts and to a lesser extent in the uteri were stimulated by P4 treatment for 3 to 4 days after ovulation. Morphology of ciliated cells was unaffected. The subtle changes did not fully account for P4-induced embryo mortality in vivo.     

Improved in vitro development of porcine embryos with different energy substrates and serum

The effect of replacing 5.5 mM glucose in North Carolina State University (NCSU)-23 medium with 0.5 mM pyruvate/5.0 mM lactate on porcine IVF embryo development was investigated in Experiment 1. Culturing embryos with pyruvate/lactate for 7 days or with pyruvate/lactate from Days 0 to 2, and then glucose from Days 2 to 7 improved cleavage rates. In Experiment 2, embryos were cultured for 7 days in pyruvate/lactate containing NCSU-23 medium supplemented with 0.05% PVA, 0.4% BSA or 10% fetal bovine serum (FBS). The BSA supplement increased the rates of cleavage, blastocyst formation, and the number of total cells in blastocysts. In Experiment 3, embryos were cultured in pyruvate/lactate containing NCSU-23 medium supplemented with 0.4% BSA for 7 days (BSA-PL), 0.4% BSA from Days 0 to 4 and then 10% FBS from Days 4 to 7 (BSA-PL-->F ) or 0.4% BSA from Days 0 to 7 with addition of 10% FBS (BSA-PL + F ) at Day 4. More blastocysts in BSA-PL--> F and hatching or hatched blastocysts in BSA-PL-->F and BSA-PL+F were obtained. Total cell number in blastocysts derived from BSA-PL-->F and BSA-PL+F were increased. Our results demonstrated that supplementing pyruvate/lactate containing NCSU-23 medium with 0.4% BSA for 4 days and replacing it with 10% FBS for another 3 days improved porcine IVF embryo development.     

Developmental responses of 2-cell embryos to oxygen tension and bovine serum albumin in Wistar rats.

To improve rat embryo culture conditions, responses of Wistar 2-cell embryos from 2 breeders to oxygen tension (5 vs 20%) and bovine serum albumin (BSA) (0 vs 3 mg/ml) were examined using rat 1-cell embryo culture medium (mR1ECM). Supplementation of 3 mg/ml BSA significantly stimulated and accelerated development to the blastocyst and expanded blastocyst stages during 72 and 96 h culture, while reduced oxygen tension stimulated cell division. Fetus development after transfer of blastocysts obtained from 72 h culture under 5% O2 with BSA was significantly higher than those cultured under atmospheric oxygen without BSA. However, the nuclear numbers of in vitro cultured blastocysts and fetus development after embryo transfer were still significantly lower than in vivo developed blastocysts, indicating the current culture condition is still suboptimal.     

Bovine blastocyst development in vitro: timing, sex, and viability following vitrification

Selection of blastocysts based on their morphological characteristics and rate of development in vitro can skew the sex ratios. The aim of this study was to determine whether an embryo's developmental rate affects its survival after vitrification, and whether male and female embryos survive vitrification differently. In vitro fertilized bovine oocytes were cultured in potassium simplex optimized medium (KSOM) + 0.1% BSA for 96 h, and then into KSOM + 1% BSA (KSOM) or in sequential KSOM + 0.1% BSA for 96 h, and then into synthetic oviduct fluid (SOF) + 5% FBS (KSOM-SOF). In part 1 of this study, embryos cultured in each medium that had developed into blastocysts at approximately 144, 156, or 180 h were recovered from culture, graded, and then vitrified. After warming, blastocyst survival rates were immediately evaluated by reexpansion of the blastocoels. In the second part of the study, all blastocysts (n = 191) were sexed by polymerase chain reaction 48 h after warming. When cultured in KSOM medium, more 144-h blastocysts survived vitrification (68%) than blastocysts vitrified at 180 h (49%). Blastocysts derived at 156 h in KSOM-SOF survived vitrification better (87%) than blastocysts vitrified at either 144 h or 180 h, and subsequently hatched at a greater rate than those vitrified at 180 h. The overall blastocyst survival rates did not differ significantly whether embryos were cultured in KSOM or sequential KSOM-SOF. Blastocysts derived at 144 and 156 h in KSOM or KSOM-SOF were predominately male, and significantly more of them survived vitrification 48 h after warming. However, blastocysts cultured in KSOM-SOF, and then vitrified at 180 h were predominately female. Overall, blastocysts that survived vitrification, and subsequently hatched 48 h after warming, were male. In summary, embryos that reached the blastocyst stage earlier were predominantly males; these males had better morphology, endured vitrification, and subsequently hatched at a greater rate than did female blastocysts.     

Correlation between diameter and DNA or protein synthetic activity in rabbit blastocysts

Noninvasive parameters are desirable to assess viability of preimplantation embryos. The objective of the present study was to investigate how noninvasive morphometric criteria are related to invasive metabolic parameters. In Day 4 and 5 noncultured and Day 4 in vitro-cultured rabbit blastocysts, diameters as well as DNA or protein synthesis (by incorporation of tritiated precursors) were measured. From the diameter of the blastocyst, total volume of embryonic cells was calculated and used for statistical analysis. In noncultured controls, cellular volume and thymidine, leucine, or methionine incorporation were highly correlated, with coefficients of correlation ranging between 0.7 and 0.9. The calculated equations of regression were linear. Blastocysts cultured for 24 or 48 h in medium supplemented with uterine flushings showed comparable coefficients of correlation and regression. After culture in serum-supplemented medium, however, a less close relationship was found, with statistically significant lower coefficients of correlation and regression. Our results demonstrate the following: (1) There is a close relationship between blastocyst diameter and metabolic criteria in noncultured rabbit blastocysts, indicating that simple measurement of the diameter of a useful tool for assessment of blastocyst metabolic activity. (2) In cultured blastocysts, however, measurement of diameter is of doubtful validity due to a substantially altered embryonic metabolism in vitro. (3) Blastocysts cultured in medium that contained uterine flushings maintained normal expansion and metabolic activity for some time.     

In utero and in vitro proteinase activity during the Mesocricetus auratus embryo zona escape time window

The goal of the present study was to investigate proteinase activity in uterine flushates collected during the zona loss time window (68-80 h post-egg activation) in both pregnant and pseudopregnant hamsters and in culture medium conditioned by hatching blastocysts. Several prominent enzyme activities appeared in all pregnant and pseudopregnant uterine flushates. However, only a 45, 43 x 10(-3) M:(r) doublet coincided with the zona loss time window; these bands were absent outside of this time window and were not found in conditioned medium. In medium conditioned by hatching blastocysts, enzyme activity was represented by a 70, 65 x 10(-3) M:(r) doublet identical to a doublet seen in all uterine flushates collected and in serum. There were 12 pregnant and 8 pseudopregnant uterine flushates that were capable of zona lytic activity in vitro (positive bioassays). Of these positive bioassays, five pregnant and four pseudopregnant uterine flushates exhibited the 45, 43 x 10(-3) M:(r) doublet (correlative positive bioassays). These data suggest that there is an important uterine contribution to blastocyst escape from the zona pellucida, consisting of proteinases secreted during a finite time window prior to blastocyst attachment that are different from the proteinases responsible for the zona lytic activity in vitro.     

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.