Effect of leukemia inhibitory factor on bovine embryos produced in vitro under chemically defined conditions

The objective of these experiments was to assess putative embryotrophic effects of leukemia inhibitory factor (LIF) on bovine preimplantation development in chemically defined media. Recombinant human LIF was added to embryo culture media at a concentration of 100 ng/ml. When added for culture of morulae LIF had no positive effect on the proportion of embryos reaching the blastocyst stage. However, LIF significantly reduced development to the blastocyst stage when added for culture of 4-cell stage embryos (P<0.05). In contrast, a positive effect was found for progression of blastocyst development. In vitro blastocyst hatching rates were significantly improved in the presence of LIF (P<0.02). Number of total cells and of inner cell mass (ICM) cells were increased in LIF-treated blastocysts. In vitro survival of frozen-thawed blastocysts was not improved by adding LIF to morula stage embryos before cryopreservation. The pregnancy rate after direct transfer of cryopreserved LIF-treated embryos was not different from that for untreated control embryos. Data indicate that addition of LIF has no major beneficial effect on bovine embryos produced in these chemically defined conditions.     

Relationship between timing of development,morula morphology,and cell allocation to inner cell mass and trophectoderm in in vitro-produced bovine embryos

Noninvasive measurements of bovine embryo quality, such as timing of cleavage, morula morphology, blastocyst formation, and hatching ability, were linked with the number of inner cell mass (ICM) cells and trophectoderm (TE) cells of the resulting embryos. First, it was confirmed that fast-cleaving embryos proved to have significantly higher chances to reach advanced developmental stages vs. intermediate and slow cleavers (P = 0.01). They also showed significantly less fragmentation at the morula stage, implying the presence of more excellent morulae among fast-cleaving embryos (P < 0.05). Second, the quality of hatched blastocysts, resulting from morulae of different morphological grades, was examined by differential staining. The total cell and ICM cell numbers were significantly lower for hatched blastocysts developed from poor morulae compared to hatched blastocysts developed from excellent, good, or fair morulae. However, hatched blastocysts with <10 ICM cells were seen in embryos belonging to all four morphological scores. Finally, it was found that timing of first cleavage was not significantly correlated with timing of blastocyst formation or with cell number of blastocysts. Timing of blastocyst formation, however, was significantly correlated with cell number: day 8 blastocysts had significantly lower total cell and ICM cell numbers than day 6 and day 7 blastocysts (P < 0.001). These results suggest that the quality of in vitro-produced bovine embryos is very variable and cannot be linked with a single criterion such as embryo morphology and/or hatching ability. Timing of blastocyst formation was the most valuable criterion with regard to embryonic differentiation. Mol. Reprod. Dev. 47:47–56, 1997. © 1997 Wiley-Liss, Inc.     

A comparative investigation on the potency of cells from the inner cell mass and trophectoderm of mouse blastocysts to produce chimeras

The ability of trophectoderm (TE) cells to produce chimeric mice (pluripotency) was compared with that of inner cell mass (ICM) cells. TE and ICM cells of blastocysts and hatching or hatched blastocysts derived from albino mice (CD-1, Gpi-1a/a) were aggregated with zona cut 8- to 16-cell stage embryos or injected into the blastocoele from non-albino mice (C57BL/6 x C3H/He, Gpi-1b/b). After transfer to pseudopregnant female mice, the contribution of the donor cells was examined by glucose phosphate isomerase (GPI) analysis of embryos, membrane and placenta at mid-gestation (Day 10.5 and 12.5) or by the coat color of newborn mice. In contrast to ICM cells, there was no contribution of TE cells in the conceptuses and no coat color chimeric young were obtained. After pre-labeling of TE cells with fluorescent latex microparticles, they were aggregated with embryos and the allocation of TE cells at the compacted morula and blastocyst stages was observed under a fluorescent microscope. Although the TE cells were observed attached onto the surface of the embryos at morula and blastocyst stages, unlike the ICM cells, they were not positively incorporated into the embryos. Thus, the pluripotency of TE cells from mouse blastocysts was not induced by the aggregation and injection methods.     

Requirement of Leukemia Inhibitory Factor or Epidermal Growth Factor for Pre-Implantation Embryogenesis via JAK/STAT3 Signaling Pathways

Leukemia inhibitory factor (LIF) plays a key role in the survivability of mouse embryos during pre-implantation. In this study, we verified the role of LIF by detecting gene expression in morula stage embryos through DNA microarray. Our results showed that LIF knockdown affected expression of 369 genes. After LIF supplementation, the epidermal growth factor (EGF) is most affected by LIF expression. To observe the correlation between LIF and EGF, the LIF knockdown embryos were supplemented with various growth factors, including LIF, EGF, GM-CSF, TGF, and IGF II. Only LIF and EGF caused the rate of blastocyst development to recover significantly from 52% of control to 83% and 93%, respectively. All of the variables, including the diameter of blastocysts, the number of blastomeres, and cells in ICM and TE, were almost restored. Moreover, EGF knockdown also impaired blastocyst development, which was reversed by LIF or EGF supplementation. The treatment with various signaling suppressors revealed that both EGF and LIF promoted embryonic development through the JAK/STAT3 signaling pathway. These data suggest that the EGF and LIF can be compensatory to each other during early embryonic development, and at least one of them is necessary for sustaining the normal development of pre-implantation embryos.     

Betaine Homocysteine Methyltransferase Is Active in the Mouse Blastocyst and Promotes Inner Cell Mass Development

Methyltransferases are an important group of enzymes with diverse roles that include epigenetic gene regulation. The universal donor of methyl groups for methyltransferases is S-adenosylmethionine (AdoMet), which in most cells is synthesized using methyl groups carried by a derivative of folic acid. Another mechanism for AdoMet synthesis uses betaine as the methyl donor via the enzyme betaine-homocysteine methyltransferase (BHMT, EC 2.1.1.5), but it has been considered to be significant only in liver. Here, we show that mouse preimplantation embryos contain endogenous betaine; Bhmt mRNA is first expressed at the morula stage; BHMT is abundant at the blastocyst stage but not other preimplantation stages, and BHMT activity is similarly detectable in blastocyst homogenates but not those of two-cell or morula stage embryos. Knockdown of BHMT protein levels and reduction of enzyme activity using Bhmt-specific antisense morpholinos or a selective BHMT inhibitor resulted in decreased development of embryos to the blastocyst stage in vitro and a reduction in inner cell mass cell number in blastocysts. The detrimental effects of BHMT knockdown were fully rescued by the immediate methyl-carrying product of BHMT, methionine. A physiological role for betaine and BHMT in blastocyst viability was further indicated by increased fetal resorption following embryo transfer of BHMT knockdown blastocysts versus control. Thus, mouse blastocysts are unusual in being able to generate AdoMet not only by the ubiquitous folate-dependent mechanism but also from betaine metabolized by BHMT, likely a significant pool of methyl groups in blastocysts.     

Morphological development,cell number,and allocation of cells to trophectoderm and inner cell mass of in vitro fertilized and parthenogenetically developed buffalo embryos: The effect of IGF-I

The morphology and number of cells in the trophectoderm (TE) and inner cell mass (ICM) of buffalo blastocysts derived from in vitro fertilization and cultured in the presence or absence of insulin-like growth factor-I (IGF-I) were analyzed by differential fluorochrome staining technique. The total cell number (TCN), TE number, and ICM cell number were significantly higher in blastocysts developed in vitro in the presence of IGF-I as compared to blastocysts developed without IGF-I (P < 0.01). It was observed that the buffalo blastocyst took 5–9 days postfertilization to develop in vitro. In order to correlate the time required for blastocyst development and the allocation of cells to TE and ICM, blastocysts were designated as fast (developing on or before day 7) or slow (developing after day 7). The TCN, TE, and ICM cells of fast-developing blastocysts cultured in the presence of IGF-I were significantly higher than slow-developing blastocysts (P < 0.01). The blastocysts developed on day 6 had a mean total cell number 118.6 ± 21.4, which significantly decreased to 85.6 ± 17.4, 62.0 ± 14.5, and 17.0 ± 4.0 on days 7, 8, and 9, respectively (P < 0.05). Normal development of buffalo embryo showed that, on average, embryos reached compact morula stage at the earliest between days 4.5–5.5. Blastocysts developed, at the earliest, between days 5.0–6.0, and it took them, on average, 6.5 days to hatch from the zona pellucida. TCN, TE, and ICM increased three times from morula to blastocyst; however, the proportion of ICM to TCN remained the same, in both embryonic stages. TE approximately doubled in hatched blastocysts, as compared to unhatched blastocysts (P < 0.05). However, ICM cells were decreased. The time required for development of parthenogenetic blastocysts was observed to be greater as compared to in vitro fertilized (IVF) blastocysts. The total cell number of parthenogenetic blastocysts was 100.8 ± 11.3, including 59.2 ± 8.4 cells of TE and 42.1 ± 6.9 cells of ICM. © 1996 Wiley-Liss, Inc.     

小鼠植入前胚胎GCN5和HDAC1表达模式及体外培养对其表达的影响

为探讨小鼠植入前胚胎组蛋白乙酰化酶GCN5(general control of nucleotide synthesis,GCN5) 和组蛋白去乙酰化酶1(histone deacetyluse1,HDAC1)的表达模式及常规体外培养对它们表达的影响,应用荧光免疫细胞化学技术,检测了体内和体外培养的小鼠2、4、8细胞期卵裂胚胎、桑葚胚和囊胚GCN5和HDAC1的表达。结果显示,GCN5在体内组各细胞期卵裂胚胎和桑葚胚的细胞浆内均呈高表达,细胞核内未见明显表达,而囊胚细胞的细胞浆和细胞核内均无表达:HDAC1在体内组小鼠2细胞期胚胎中以细胞浆内表达为主,在其他各期胚胎均以细胞核内表达为主．囊胚期内细胞团部分细胞的细胞核内未见HDAC1表达。GCN5在体外组小鼠植入前各期胚胎均不表达,而 HDAC1的表达强度明显低于体内组的。提示体外培养抑制小鼠植入前胚胎GCN5和明显降低 HDAC1的表达,影响胚胎基因的正确性表达。     

小鼠植入前胚胎GCN5和HDAC1表达模式及体外培养对其表达的影响

为探讨小鼠植入前胚胎组蛋白乙酰化酶GCN5（general control of nucleotide synthesis,GCN5）和组蛋白去乙酰化酶1（histone deacetylasel,HDAC1）的表达模式及常规体外培养对它们表达的影响,应用荧光免疫细胞化学技术,检测了体内和体外培养的小鼠2、4、8细胞期卵裂胚胎、桑葚胚和囊胚GCN5和HDAC1的表达。结果显示,GCN5在体内组各细胞期卵裂胚胎和桑葚胚的细胞浆内均呈高表达,细胞核内未见明显表达,而囊胚细胞的细胞浆和细胞核内均无表达：HDACl在体内组小鼠2细胞期胚胎中以细胞浆内表达为主,在其他各期胚胎均以细胞核内表达为主。囊胚期内细胞团部分细胞的细胞核内未见HDAC1表达。GCN5在体外组小鼠植入前各期胚胎均不表达。而HDAC1的表达强度明显低于体内组的。提示体外培养抑制小鼠植入前胚胎GCN5和明显降低HDAC1的表达,影响胚胎基因的正确性表达。     

Characterization of embryos derived from calf oocytes: kinetics of cleavage, cell allocation to inner cell mass, and trophectoderm and lipid metabolism

Embryos derived from calf oocytes were compared with adult cow oocyte-derived embryos (1) by studying the kinetics of embryo development using time-lapse cinematography (2) by evaluating the ratio between inner cell mass (ICM) and trophectoderm (TE) cells in blastocysts (3) by measuring the triglyceride content of the blastocysts. The rate of calf oocyte-derived embryos reaching the blastocyst stage was reduced (26 vs. 46% for adult derived embryos). Calf oocyte-derived embryos preferably arrested their development before the 9-cell stage. Those that developed into blastocysts had cleaved earlier to reach the 2-cell or 3-cell stages than embryos that arrested before the 9-cell stage. The 9-cell stage tended to appear later in calf oocyte-derived embryo that reached the blastocyst stage than in adult-derived embryos. This difference became significant at the morula stage. Accordingly, the fourth cell cycle duration was longer for calf oocyte-derived embryos. Day 8 blastocysts from both sources had similar total cell numbers (calf: 89 +/- 20; cow: 100 +/- 30) and cell distribution between TE and ICM. The triglyceride content of day 7 blastocysts was similar for both sources (64 +/- 15 vs. 65 +/- 6 ng/embryo, respectively). In conclusion, calf oocyte-derived embryos are characterized by a higher rate of developmental arrest before the 9-cell stage and by a longer lag phase preceding the major onset of embryonic genome expression. These changes might be related to insufficient "capacitation" of the calf oocyte during follicular growth. Despite these differences, modifications in the quality of the resulting blastocysts were not detected.     

Cell allocation to the inner cell mass and the trophectoderm in rat embryos during in vivo preimplantation development

Summary The number of trophectoderm (TE) and inner cell mass (ICM) cells was determined by complementmediated lysis and differential staining in rat embryos collected at different times during in vivo preimplantation development. At 90 h after fertilization, two groups of morulae were discriminated according to the presence or absence of detectable ICM cells, and the analysis of their total cell number indicated that acquisition of a permeability seal between TE cells begins at the 14-cell stage. On the other hand, our data confirmed that blastocoele formation occurs after the fourth cleavage division in the rat. The total cell number increased exponentially with time in blastocysts recovered between 90 h and 127 h but the cell kinetics of TE and ICM cells were different. The proportion of ICM cells consequently varied throughout blastocyst development, with a peak value for expanded blastocysts at 103 h. Finally, a linear-quadratic relationship was found between the numbers of TE and ICM cells when all the embryos with a detectable ICM were analysed together.     

3H-uridine incorporation in early porcine embryos.

The present study investigated the ontogeny of 3H-uridine incorporation into RNA as a measure for RNA synthesis in preimplantation porcine embryos from the two-cell stage up to the stage of the newly hatched blastocyst. A total of 568 embryos were cultured in vitro for 3 hr in medium (KRB plus lamb serum) containing 9 microM 3H-uridine. After disruption of cell membranes, RNA was isolated on DEAE cellulose filters, and the radioactivity was taken as a measure for the rate of RNA synthesis. No RNA synthesis was detected at the two-cell stage. From the four-cell to the morula stage, 3H-uridine incorporation per embryo increased about ninefold (P less than 0.001); in blastocyst stages, the increase between developmental stages was not statistically significant. Hatched blastocysts had the highest genomic activity. On a per cell basis, 3H-uridine incorporation was not different from the four-cell stage up to the zona pellucida-intact blastocyst and amounted to 0.29-0.37 fmol 3H-uridine incorporation/cell/3 hr. In hatched blastocysts, 3H-uridine incorporation per blastomere was increased (P less than 0.01 compared with younger stages) and amounted to 0.86 fmol 3H-uridine incorporation/cell/3 hr. It is concluded that 1) the rate of uridine incorporation depends on the cell stage in zona pellucida-intact porcine embryos and 2) uridine incorporation per blastomere is significantly increased in hatched blastocysts compared with earlier stages.     

A paucity of structural integrity in cloned porcine blastocysts produced in vitro

The structural integrity of blastocyst stage embryos, consisting of the inner cell mass (ICM) and trophectoderm (TE) cells, is a prerequisite for normal development after implantation in mammals. In this study, allocation of nuclear transfer (NT)-derived porcine blastocysts to the ICM and to the TE cells was examined and compared with IVF- and in vivo-derived embryos. NT-derived embryos had a lower developmental competence to the blastocyst stage than IVF-derived embryos (P < 0.05). Total cell number of NT-derived blastocysts was inferior to that of IVF-derived embryos (P < 0.05), although no difference was detected between the two groups in the ratio of ICM to total cells. However, in vivo-derived blastocysts had a higher proportion of ICM to total cells compared with in vitro-produced embryos (P < 0.01). To investigate what proportions of in vitro-produced porcine embryos represent normal structural integrity, differentially-stained blastocysts were individually classified into three presumptive groups (I: <20%; II: 20-40%; III: >40%) according to the ratio of ICM to total cells. Low proportions of NT- (12.5%, 7/56) and IVF-derived blastocysts (15.8%, 9/57) were assigned to Group II, presumptively having a normal range of structural integrity, whereas, almost all in vivo-derived embryos (97.5%, 39/40) were allocated to Group II. In conclusion, limited structural integrity may lead to the poor survival to term of NT- or IVF-derived porcine embryos produced in vitro.     

Expression of a male-specific factor on various stages of preimplantation bovine embryos

Four-cell to blastocyst stage bovine embryos were collected from superovulated donors and cultured for 90 min in Ham's F-10 medium (HF-10) containing 10% (V/V) absorbed anti-histocompatibility (H)-Y antiserum. Embryos were then washed 3 times and placed in HF-10 supplemented with 10% (V/V) fluorescein isothiocynate (FITC)-conjugated goat anti-mouse gamma globulin. After an additional wash, embryos were placed in fresh drops of HF-10, individually evaluated at 200 X magnification, and classified as either fluorescent (H-Y-positive) or nonfluorescent (H-Y-negative). Embryos were then placed in drops of HF-10 containing 14% vinblastin and cultured for 4-6 h. Embryos were coded and individually karotyped, and the sex chromosomes were identified. H-Y antigen was detected as early as the eight-cell stage, but not at the four-cell stage. Seventy-nine percent of fluorescent embryos and 89% of nonfluorescent embryos were XY and XX, respectively. Another experiment was carried out in which H-Y antigen was detected on intact inner cell masses (ICM) isolated by immunosurgery from expanded blastocysts that also had been assayed for H-Y antigen. Eighty-eight and 92%, respectively, of ICM classified as fluorescent or nonfluorescent had been scored the same as intact blastocysts. It is concluded from these data that H-Y antigen can be detected on eight-cell to blastocyst stage bovine embryos. There appears to be a localization of detectable antigen in the area of the ICM at the expanded blastocyst stage. Detection of H-Y antigen is an effective, noninvasive method for identification of the sex of preimplantation bovine embryos.     

Stage-specific response of preimplantation mouse embryos to W-7, a calmodulin antagonist

Involvement of calmodulin-dependent processes in preimplantation development of mouse embryos was studied with the use of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a specific antagonist of calmodulin. At 25 microM, W-7 interfered with compaction of eight-cell embryos, caused decompaction of compacted eight-cell embryos, inhibited cavitation of late morulae, and caused collapse and degeneration of blastocysts. These effects of W-7 appear to be due to specific inhibition of calmodulin-dependent processes, because W-5, a less active analogue of W-7, was less effective in interfering with development; at 25 microM, W-5 had only a slight effect on compaction and had no effect on blastocyst formation, maintenance of blastocoels, or post-blastocyst development. In addition to the developmental effects just described, W-7 inhibited cell proliferation in four-cell embryos and reduced cell numbers of morulae after treatment at the two- to eight-cell stages. There was a marked increase in embryos' sensitivity to W-7 at the late morula stage, and the sensitivity increased further as embryos developed into blastocysts; the effects of W-7 were largely reversible after treatment at the two-cell through the compacted eight-cell stages, but not after treatment at the late morula or blastocyst stage. At the blastocyst stage, inner cell mass cells appeared to be slightly more resistant to W-7 than trophectoderm cells. This differential sensitivity became more pronounced at the late blastocyst stage: after 3.5-4-h exposure of late blastocysts to 25 microM W-7, all trophectoderm cells degenerated but most of the inner cell masses survived. From these results it appears that calmodulin-dependent processes are involved in development of mouse embryos at all of the preimplantation stages examined.     

Metabolism and cell allocation during parthenogenetic preimplantation mouse development

Diploid parthenogenetic postimplantation mouse embryos, containing two maternal genomes, are characterized by poor development of extraembryonic membranes derived from the trophectoderm and primitive endoderm of the blastocyst. This is thought to be caused by a deficiency of expression of paternally derived imprinted genes. Here we have compared the inner cell mass, from which the primitive endoderm and fetal lineages are derived, and the trophectoderm, which forms a major component of the placenta, in parthenogenetic and fertilized preimplantation embryos. We have also studied the metabolism from the 1-cell to the blastocyst stage. Cell numbers were reduced in the ICM and TE of parthenogenetic blastocysts compared to fertilized blastocysts. This was thought to be due to the increased levels of cell death observed in these lineages. Pyruvate and glucose uptake by parthenogenetic embryos was similar to that by fertilized embryos throughout preimplantation development. However, at the expanded blastocyst stage glucose uptake by parthenogenetic embryos was significantly higher than by fertilized embryos. The implications of the actions of imprinted genes and of X-inactivation is discussed. © 1996 Wiley-Liss, Inc.     

Enhancement of radiation effects by mercury in early postimplantation mouse embryos in vitro

Summary Two-cell mouse embryos were X-irradiated (1 Gy) and immediately thereafter exposed to mercuric chloride (3 µM) up to the blastocyst stage. When combined treatment started shortly (about 1 to 2 h) before mitosis to the four-cell stage, blastocyst formation, hatching of blastocysts, trophoblast outgrowth and ICM formation were impaired stronger than expected from the addition of the single effects. The enhancement of risk was maximal for hatching of blastocysts and no further increase was observed for trophoblast outgrowth and ICM formation. When exposure of embryos to X-rays and mercury began about 5 to 6 h before mitosis to the four-cell stage, only additive effects were obtained for the endpoints mentioned above.     

Intercellular communication in in vivo- and in vitro-produced bovine embryos.

In vivo bovine embryos were obtained by nonsurgical flushing of uterine horns of cows submitted to superovulatory treatment, while in vitro embryos were generated from oocytes collected from slaughtered donors. Lucifer Yellow injected into single blastomeres did not diffuse into neighboring cells until the morula stage in in vivo embryos and the blastocyst stage in in vitro embryos. In both cases diffusion was limited to a few cells. In contrast, diffusion was extensive in microsurgically isolated inner cell mass (ICM) but absent in the trophectoderm (TE). At the blastocyst stage, diffusion was always more extensive in in vivo than in in vitro embryos. Ultrastructural analyses confirmed these functional observations, and gap junction-like structures were observed at the blastocyst stage. These structures were diffuse in the ICM of in vivo embryos, scarce in the ICM of in vitro embryos and in the TE of in vivo embryos, and not observed in the TE of in vitro embryos. Blastomeres at all stages of development from the 2-cell stage to the blastocyst stage in in vitro embryos and at the morula and blastocyst stage in in vivo embryos were electrically coupled, and the junctional conductance (Gj) decreased in in vitro embryos from 4.18 +/- 1.70 nS (2-cell stage) to 0.37 +/- 0.12 nS (blastocyst stage). At each developmental stage, in vivo embryos showed a significantly (P < 0. 05) higher Gj than in vitro-produced embryos. Moreover, a significantly (P < 0.01) higher Gj was found in isolated ICM than in the respective blastocyst in both in vivo- and in vitro-produced embryos (3.5 +/- 1.4 vs. 0.7 +/- 0.3 and 2.6 +/- 1.6 vs. 0.37 +/- 0. 12 nS, respectively). The electrical coupling in absence of dye coupling in the early bovine embryo agrees with observations for embryos from other phyla. The late and reduced expression of intercellular communicative devices in in vitro-produced embryos may be one of the factors explaining their developmental low efficiency.     

Localization and binding of transforming growth factor-beta isoforms in mouse preimplantation embryos and in delayed and activated blastocysts.

The stage and cell-specific accumulation of mammalian isoforms of transforming growth factor-beta (TGF-beta 1, TGF-beta 2, and TGF-beta 3) and TGF-beta binding were examined in the preimplantation embryo and in progesterone (P4)-treated delayed or P4 plus estradiol-17 beta (E2)-treated activated blastocysts in the mouse. Immunocytochemical studies revealed that while all three immunoreactive TGF-beta isoforms were present in one-cell embryos, very little or no immunostaining was observed in two-cell embryos. However, distinct immunostaining of these isoforms was again observed in four-cell embryos and persisted through the blastocyst stage. Among the isoforms studied, TGF-beta 2 immunostaining showed a unique pattern in late morulae. In many of these morulae, the staining was primarily observed in outside cells. However, in blastocysts, immunostaining for all three isoforms was present both in the inner cell mass (ICM) and trophectoderm (Tr). Immunostaining in sectioned blastocysts and immunosurgically isolated ICMs confirmed immunostaining in Tr and ICM cells. To ascertain whether preimplantation embryos can produce TGF-beta isoforms, immunostaining was performed in embryos grown in vitro from two-cell stage in simple balanced salt solution. Immunoreactive TGF-beta s 1-3 were present in embryos at all stages of development examined (four-cell embryos through blastocysts). The virtual absence of immunoactive TGF-beta s in two-cell embryos but their accumulation in embryos at later stages of development in vitro provides evidence that these growth factors were produced by embryos. In order to assess at what stages of development preimplantation embryos could be responsive to TGF-beta s, specific binding of [125I]TGF-beta 1 and [125I]TGF-beta 2 was performed in embryos and examined by autoradiography. Low levels of binding were first detected in eight-cell embryos. The binding increased in morulae followed by a further increase in blastocysts. Analysis of binding of [125I]TGF-beta 2 in immunosurgically isolated ICMs indicated that binding was primarily evident in Tr cells. Affinity labeling of TGF-beta 1 or TGF-beta 2 in Day 4 blastocysts revealed three classes of binding proteins with approximate molecular sizes of 65 kDa (type I), 90 kDa (type II), and greater than 250 kDa (type III), in addition to a doublet of 130 and 140 kDa proteins. This observation is similar to those reported for other cell types. The data suggest that embryos are likely to be responsive to TGF-beta s after the third cleavage.(ABSTRACT TRUNCATED AT 400 WORDS)