In vitro development of bovine one-cell embryos: Influence of glucose, lactate, pyruvate, amino acids and vitamins

To elucidate the effect of nutrient substrates on embryo development, in vitro fertilized bovine one-cell embryos were cultured in a medium similar to synthetic oviduct fluid (SOF) but without glucose and containing 3.3 mM lactate, 0.3 mM pyruvate and 3 mg/ml bovine serum albumin (BSA) at 39 degrees C in 5% CO(2) in air. Results indicated that addition of glucose was not only unnecessary, but it also had a deleterious effect on embryo development to the morula stage. Lactate supported embryo development up to the morula stage as well as pyruvate. Supplementation with 20 amino acids contained in basal medium Eagle's (BME) and minimum essential medium (MEM) improved development to the morula stage dramatically and increased the cell number compared with that of the controls. Addition of the vitamins from MEM to SOF had no beneficial effect. The SOF with amino acids did not increase the frequency of blastocysts 7 days after in-vitro fertilization but did increase the total number of cells compared with that of the controls. Frequency of blastocysts at Day 7 in SOF with amino acids was equivalent to that of co-culture although the total cell number was lower. These results demonstrate that a semi-chemically defined medium can successfully support the development of bovine embryos to the morula stage to a limited extent, but the medium lacks some nutrients or growth factors to fully support development through the blastocyst stage.     

The influence of insulin on the in vitro development of mouse and bovine embryos

To further investigate the role of insulin during preimplantation embryo development, we compared the effects of insulin on the development of mouse and bovine preimplantation embryos and on cell proliferation during culture in vitro in simplex media. The influence of insulin on the development of mouse zygotes was determined during cultivation in mSOF medium, alone or supplemented with glucose. Similarly, the effects of insulin on the bovine preimplantation embryo development were studied in mSOF medium. The addition of insulin into mSOF medium enhanced significantly the number of cells per mouse blastocyst. Moreover, when mSOF medium was supplemented with insulin and 0.2 mmol x l(-1) glucose, the percentage of hatched blastocysts and the mean cell number of mouse blastocysts were significantly higher. Insulin had no significant effect on the development of bovine embryos, produced by in vitro fertilization of in vitro matured oocytes. Neither the rates of developing embryos nor the mean number of cells in blastocysts were different in comparison with control embryos. Our results suggest that the in vitro development of mouse embryos could be enhanced by the addition of insulin to the culture medium and is further improved by the addition of glucose. In contrast to this our results indicate that insulin has no detectable beneficial effect on the preimplantation development of bovine embryos in mSOF medium.     

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.     

Inhibitory effect of IGF-I on induced apoptosis in mouse preimplantation embryos cultured in vitro

Insulin-like growth factor I (IGF-I) has been shown to promote mammalian early embryo development. Increased cell division or decreased cell death have been proposed as two main possible mechanisms in its effect. Here we examine the nature of this promoting effect in a model situation. Camptothecin (0.01 microg/ml) and actimomycin D (0.005 microg/ml) were used to induce apoptosis. Four-cell mouse embryos were cultured in vitro to blastocyst stage in the temporary (15 h) presence or absence of apoptotic inductors and in the permanent presence or absence of IGF-I (100 ng/ml). Embryos were assessed by morphological triple staining (Hoechst 33342, propidium iodide, Calcein AM) and comet assay on Day 5, 120 h after administration of hCG. The number of nuclei, the blastocyst formation, the proportion of embryos containing fragmented DNA and the percentage of apoptotic and secondary necrotic nuclei were assessed. Both inductors of apoptosis significantly increased the percentage of apoptotic and secondary necrotic cells and reduced total cell counts (camptothecin, P>0.001; actinomycin D, P>0.001). When IGF-I was added to the culture medium in the presence of an apoptosis inductor, apoptosis incidence was significantly decreased (P<0.001). The addition of IGF-I into control samples also decreased the percentage of apoptotic and secondary necrotic cells. In contrast, IGF-I addition had no significant influence on embryo development (P>0.05). Our data suggest a primary role for IGF-I as an apoptotic survival factor in mouse preimplantation embryos in specific conditions.     

The role of IGF-I, cAMP/protein kinase A and MAP-kinase in the control of steroid secretion, cyclic nucleotide production, granulosa cell proliferation and preimplantation embryo development in rabbits

The aim of this study was to investigate the actions of insulin-like growth factor I (IGF-I) on the secretory and proliferative functions of rabbit ovarian cells and on early embryogenesis. It was found that addition of IGF-I at a lower concentration (1 ng/ml) stimulated progesterone secretion by cultured rabbit granulosa cells, whilst higher concentrations of IGF-I (10, 100 ng/ml) were inhibitory. IGF-I had no effect on estradiol secretion. Cyclic AMP secretion was slightly increased after addition of IGF-I at 10 ng/ml, but not by higher concentrations. Cyclic GMP secretion was stimulated by IGF-I at 100 ng/ml only. A blocker of protein kinase A, Rp-cAMPS, did not alter progesterone and estradiol secretion but did prevent the action of IGF-I on progesterone secretion. An immunocytochemical study demonstrated that IGF-I significantly increased the proportion of proliferating cell nuclear antigen-positive (PCNA-positive) cells. Rp-cAMP did not change cell proliferation but partially prevented the proliferation-stimulating effect of IGF-I. IGF-I (100 ng/ml) significantly increased the proportion of divided zygotes and the number of embryos reaching the morula/blastocyst stage. Blockers of PKA, Rp-cAMPS and KT5720, reversed the effects of IGF-I on zygote cleavage and embryo development. Addition of IGF-I (100 ng/ml) significantly increased MAPK within the cells (proportion showing immunoreactivity to ERK-1 and ERK-3 antibodies and intensity of a 42 kDa band related to ERK-2). Rp-cAMPS suppressed the basal ERK-2 immunoreactivity but not that of ERK-1 or ERK-3. It completely inhibited the IGF-I-induced activation of ERK-3 but not that of ERK-1 or ERK-2. This in vitro study demonstrates that IGF-I is a potent stimulator of ovarian secretion, proliferation and embryogenesis in rabbit. Its effects are mediated by cAMP/PKA- and, probably by, MAPK-dependent intracellular mechanisms.     

Post-thaw culture in presence of insulin-like growth factor I improves the quality of cattle cryopreserved embryos

The goal of this study was to examine the effect of insulin-like growth factor I (IGF-I; added during post-thaw culture (48 h)) on the preimplantation viability and quality of cryopreserved bovine in vivo recovered embryos. The morula stage embryos, non-surgically recovered from superovulated dairy cows of Czech Fleckvieh cattle breed, had previously been cryopreserved by a slow freezing technique and stored in liquid nitrogen since 1989-1990. Following thawing, the embryos were cultured for 48 h either alone (no IGF-I) or in the presence of IGF-I (10 or 100 ng/ml); non-cultured embryos served as a control. Thereafter, the embryos were analyzed for cleavage to the blastocyst stage, apoptosis (TUNEL), embryo cell number and quality of actin cytoskeleton. Following post-thaw culture 41% of embryos developed to advanced blastocysts. IGF-I increased this per cent and, at a higher dose, essentially reduced the per cent of degenerated embryos. In cultured embryos, IGF-I at both doses elevated the cell number compared with non-cultured embryos. However, in comparison with embryos cultured without IGF-I, only the higher IGF-I dose resulted in elevating the embryo cell number. The TUNEL index was significantly lowered by IGF-I treatment. Thawed embryos were mostly of the grade III actin type and fewer (12%) had grade II actin, whilst no grade I actin embryos were noted. The addition of IGF-I resulted in the appearance of grade I actin embryos (8.33 and 6.9% for 10 and 100 ng/ml, respectively). These observations indicate that the addition of IGF-I during post-thaw culture can improve the quality of bovine cryopreserved embryos.     

Acute IGF-I infusion stimulates protein synthesis in skeletal muscle and other tissues of neonatal pigs

Studies have shown that protein synthesis in skeletal muscle of neonatal pigs is uniquely sensitive to a physiological rise in both insulin and amino acids. Protein synthesis in cardiac muscle, skin, and spleen is responsive to insulin but not amino acid stimulation, whereas in the liver, protein synthesis responds to amino acids but not insulin. To determine the response of protein synthesis to insulin-like growth factor I (IGF-I) in this model, overnight-fasted 7- and 26-day-old pigs were infused with IGF-I (0, 20, or 50 microg. kg(-1). h(-1)) to achieve levels within the physiological range, while amino acids and glucose were clamped at fasting levels. Because IGF-I infusion lowers circulating insulin levels, an additional group of high-dose IGF-I-infused pigs was also provided replacement insulin (10 ng. kg(-0.66). min(-1)). Tissue protein synthesis was measured using a flooding dose of L-[4-(3)H]phenylalanine. In 7-day-old pigs, low-dose IGF-I increased protein synthesis by 25-60% in various skeletal muscles as well as in cardiac muscle (+38%), skin (+24%), and spleen (+32%). The higher dose of IGF-I elicited no further increase in protein synthesis above that found with the low IGF-I dose. Insulin replacement did not alter the response of protein synthesis to IGF-I in any tissue. The IGF-I-induced increases in tissue protein synthesis decreased with development. IGF-I infusion, with or without insulin replacement, had no effect on protein synthesis in liver, jejunum, pancreas, or kidney. Thus the magnitude, tissue specificity, and developmental change in the response of protein synthesis to acute physiological increases in plasma IGF-I are similar to those previously observed for insulin. This study provides in vivo data indicating that circulating IGF-I and insulin act on the same signaling components to stimulate protein synthesis and that this response is highly sensitive to stimulation in skeletal muscle of the neonate.     

Influences of epidermal growth factor and insulin-like growth factor-I on bovine blastocyst development in vitro

Experiments were carried out to investigate putative beneficial effects of adding epidermal growth factor (EGF) or insulin-like growth factor-I (IGF-I) for bovine embryo culture in chemically defined media. Presumptive zygotes (18 h post-insemination) were randomly assigned to culture treatments. In experiment 1, treatments involved additions of recombinant human EGF to provide concentrations of 0 ng (control), 1, 5, and 25 ng/ml. No differences were seen in numbers of 4-cell stage embryos between groups. A concentration of 5 ng/ml EGF but not 1 or 25 ng/ml during embryo culture improved percentages of 4-cell stage embryos reaching blastocysts compared to the control (P<0.05). Numbers of inner cell mass (ICM) cells and trophoblast cells of day 8 blastocysts were similar for the control and 5 ng/ml EGF-treated groups. In experiment 2, culture with recombinant human IGF-I in concentrations of 0 ng (control), 2, 10, and 50 ng/ml resulted in no differences in numbers of 4-cell stage embryos between groups. When compared to controls, IGF-I treatments at 10 and 50 ng/ml improved proportions of 4-cell stage embryos that reached blastocysts (P<0.05). In experiment 3, numbers of ICM cells of day 8 blastocysts were significantly higher after being cultured with 50 ng/ml of IGF-I compared to those of the controls (P<0.05). No additive effect of combining EGF (5 ng/ml) and IGF-I (50 ng/ml) was seen when results were compared to those following supplementation of the media with either EGF or IGF-I alone. In conclusion, both EGF and IGF-I could independently enhance bovine preimplantational development in chemically defined media and IGF-I but not EGF may play a mitogenic role during early bovine development.     

Effects of growth hormone and insulin-like growth factor-I on development of in vitro derived bovine embryos

The objectives of this study were to determine whether the addition of growth hormone (GH) to maturation medium and GH or insulin-like growth factor-I (IGF-I) to culture medium affects development of cultured bovine embryos. We matured groups of 10 cumulus-oocyte complexes (COCs) in serum-free TCM-199 medium containing FSH and estradiol with or without 100 ng/ml GH. After fertilization, we transferred groups of 10 putative zygotes to 25 microl drops of a modified KSOM medium containing the following treatments: non-specific IgG (a control antibody, 10 microg/ml); GH (100 ng/ml) + IgG (10 microg/ml, GH/IgG); IGF-I (100 ng/ml) + IgG (10 microg/ml, IGF/IgG); antibody to IGF-I (10 microg/ml, anti-IGF); GH (100 ng/ml) + anti-IGF (10 microg/ml GH/anti-IGF); IGF-I (100 ng/ml) + anti-IGF (10 microg/ml, IGF/anti-IGF); no further additions (control). We repeated the experiment six times. Adding GH to the maturation medium increased cleavage rates at Day 3 compared to control (87.3 +/- 1.2% > 83.9 +/- 1.2%; P < 0.05) but had no effects on blastocyst development at Day 8. At Day 8, blastocyst development was greater (P < 0.01) for GH/IgG (24.8 +/- 2.5%) and IGF/IgG (33.7 +/- 2.5%) than for IgG (16.1 +/- 2.1%) and greater for IGF/IgG than for GH/IgG (P < 0.02). Blastocyst development at Day 8 did not differ between anti-IGF (20.4 +/- 1.8%) and GH/anti-IGF (24.1 +/- 1.9%) or IGF/anti-IGF (17.7 +/- 1.9%), but it was greater for GH/anti-IGF than for IGF/anti-IGF (P < 0.05). The Day 8 blastocysts of GH/IgG and IGF-I/IgG groups had a higher (P < 0.01) number of cells than the IgG group. The addition of anti-IGF-I eliminated the effects of IGF-I on cell number but did not alter GH effects. In conclusion, both GH and IGF-I stimulate embryonic development in cattle and GH effects may likely involve IGF-I-independent mechanisms.     

Antioxidant requirements for bovine oocytes varies during in vitro maturation,fertilization and development

Antioxidants may be beneficial additives to synthetic culture media because these well defined media lack serum or other macromolecules that serve as reactive oxygen species scavengers. In this study, three separate experiments were performed to determine the effects of antioxidants on the development of oocytes to the morula and blastocyst stage when added during in vitro maturation (IVM) of bovine oocytes, during in vitro fertilization (IVF), and during embryo culture for the first 72 h of the development period. Bovine oocytes were matured, fertilized (under 20% O(2)), and embryos were cultured (under 7% O(2)) in defined conditioned medium in vitro with or without supplementation with the antioxidant cysteine, N-acetyl-L-cysteine (NAC), catalase and superoxide dismutase (SOD). Significant improvements in the proportion of oocytes undergoing morula and blastocyst development (33.3% versus 20.3%, P<0.05) were achieved when cysteine (0.6 mM) was added to the maturation medium as compared to control medium without antioxidant supplementation. However, the addition of NAC (0.6mM), catalase (5 or 127 U/ml) or SOD (10 or 1000 U/ml) to the maturation medium did not improve the proportion of oocytes undergoing morula and blastocyst development. During the IVF period, addition of antioxidants (cysteine or NAC 0.6mM, catalase 127U/ml, SOD 100U/ml) significantly reduced the subsequent rate of bovine embryo development to the morula and blastocyst stage (P<0.05). In a defined medium for embryo culture (7% O(2)), the addition of cysteine improved the development of bovine embryos while NAC, catalase and SOD had no positive effect on embryonic development. Our study showed that medium supplementation with cysteine during IVM and in vitro culture (IVC) improved the rate of bovine embryo development, in contrast to extracellular antioxidants like catalase and SOD that caused no improvement.     

Development, freezability and amino acid consumption of bovine embryos cultured in synthetic oviductal fluid (SOF) medium containing amino acids at oviductal or uterine-fluid concentrations

In this study, we examined the development, freezability and amino acid consumption of in vitro produced bovine embryos cultured in a chemically defined medium (SOF+polyvinyl alcohol), supplemented with 24 amino acids at concentrations measured in bovine oviductal or uterine fluid. Amino acids at concentrations in oviductal fluid tested by Elhanssan (EOAA) significantly improved development to the hatched blastocyst stage, compared to Sigma amino acid solutions BME and MEM (SAA). Amino acids at concentrations in uterine fluid tested by Li (LUAA) were not compared to SAA, and development in LUAA was not significantly different from development in EOAA. Amino acids at concentrations in uterine fluid tested by Elhanssan (EUAA) significantly reduced cleavage rate and blocked further embryo development. When the IVF embryos were cultured in EOAA for 48, 72, 96, or 120 h and then transferred to LUAA, blastocyst and hatched blastocyst rates were not significantly affected. The freezability of blastocysts cultured in EOAA for the first 72 h and then moved to LUAA was improved compared to that in SAA. During the 1-8-cell stages, embryos secreted all 23 amino acids (total, 6,368 pmol/embryo). During the 8-cell to morula stages, embryos continued to secrete 21 amino acids (total, 2,495 pmol/embryo), meanwhile embryos began to absorb Arg (70 pmol/embryo) and Gln (18 pmol/embryo). After the morula stage, embryos began to absorb 15 amino acids including Glu, Gly, Arg, and Gln (total, 2,742 pmol/embryo) and secreted eight amino acids (total, 1,616 pmol/embryo). Embryos absorbed only Arg (183 pmol/embryo) and secreted the other 22 amino acids (total, 3,697 pmol/embryo) when the culture medium was not changed during the entire culture period (zygote to blastocyst).     

Developmental failure of hybrid embryos generated by in vitro fertilization of water buffalo (Bubalus bubalis) oocyte with bovine spermatozoa

The developmental potential of inter-species hybrid embryos produced by in vitro fertilization of in vitro matured buffalo oocytes with bovine spermatozoa was studied with a view to investigate pre-implantation embryo development and its gross morphology, early embryonic gene expression, and embryonic genome activation. Fertilization events with both buffalo and cattle spermatozoa were almost similar. Overall fertilization rate with cattle spermatozoa was 78.4% was not significantly different from that of buffalo spermatozoa (80.2%). Initial cleavage rate between buffalo and hybrid embryo was also similar, and there was no significant difference in their developmental rate till 8-cell stage (26.0 +/- 4.1 vs. 24.3 +/- 4.8). However, only 5.3% of hybrid embryos developed into blastocyst stage compared to 21.7% in buffalo. mRNA phenotyping of insulin-like growth factor family (Insulin, insulin receptor, IGF-I, IGF-I receptor, IGF-II, and IGF-II receptor) and glucose transporter isoforms (GLUT-I, II, III, IV) in hybrid embryos clearly showed that these molecules were not expressed after 8-cell stage onward. Similarly, as observed in buffalo embryos, incorporation of (35)S-methionine and (3)H-uridine could not be observed in hybrid embryos from 8-cell stage onward. This suggests that the maternal-zygotic genome activation did not occur in hybrid embryos. Differential staining also showed that the blastomere stopped dividing after 8-cell stage. Collectively, these parameters clearly showed that there was developmental failure of hybrid embryos.     

Insulin-like growth factor binding protein-3 mediates IGF-I action in a bovine mammary epithelial cell line independent of an IGF interaction

IGF-I is mitogenic for the bovine mammary epithelial cell line MAC-T. In addition, IGF-I specifically upregulates IGFBP-3 synthesis in these cells. To investigate this effect on cell growth and IGF-I responsiveness, cell lines were developed that constitutively express IGFBP-3. MAC-T cells transfected with IGFBP-3 (+BP3) or vector alone (Mock) grew similarly over 7 days in 10 or 1% fetal calf serum. Basal DNA synthesis was lower (70%) in +BP3 cells compared to Mock cells. However, DNA synthesis was increased by IGF-I (1-50 ng/ml) relative to untreated controls to a greater extent in +BP3 cells compared to Mock cells. IGF-I (20 ng/ml) increased DNA synthesis 11- and threefold in +BP3 and Mock cells, respectively. Additionally, +BP3 cells were more sensitive to the lower concentrations of IGF-I (1-5 ng/ml). In contrast, preincubation of Mock cells with exogenous IGFBP-3 did not enhance responsiveness or sensitivity to IGF-I. Basal DNA synthesis was unaffected by either an IGF neutralizing antibody or exogenous IGFBP3, indicating the differences observed between +BP3 and Mock cells were not attributable to sequestration of endogenous IGF-I by IGFBP-3. There were no differences between +BP3 and Mock cells in IGF-I receptor number or affinity. DNA synthesis was also increased in +BP3 cells, compared to controls, in response to 5 microg/ml insulin and 2.5 ng/ml Long R(3)IGF-I, indicating that the potentiated response did not require an interaction with IGFBP-3. These results suggest that IGF-I regulation of IGFBP-3 represents a regulatory loop, the function of which is to increase IGF-I bioactivity, using a mechanism that does require an IGF-I-IGFBP-3 interaction.     

TUNEL analyses of bovine blastocysts after culture with EGF and IGF-I

Experiments were carried out to investigate the beneficial effects of IGF-I or EGF on bovine embryo development in chemically defined embryo culture media and resultant incidences of nuclear DNA fragmentation as an indication of embryo quality. Presumptive IVF zygotes were randomly cultured in either control (with no added growth factor) or treatment groups, i.e., with 50 ng/ml IGF-I (experiment 1) or 5 ng/ml EGF (experiment 2). IGF-I supplemented to culture media significantly improved proportions of blastocysts from oocytes inseminated compared to untreated controls (38.0% vs. 28.5%). Only embryos reaching the blastocyst stage on day 8 showed significant effects of IGF-I treatment by resulting in higher blastocyst cell numbers (162 vs. 141) and lower percentages of TUNEL positive nuclei (2.1% vs. 3.3%) when compared to controls. Blastocyst development from oocytes was also improved by EGF supplementation compared to untreated controls (38.5% vs. 30.7%). Cell numbers of either day 7 or day 8 blastocysts were not affected by EGF treatment, nor were percentages of TUNEL positive nuclei when compared with controls. Similar proportions of parthenogenetically activated oocytes developed to blastocysts as for inseminated oocytes (28.8%). Parthenogenetic blastocysts contained fewer cells (93) and an increased percentage of TUNEL positive nuclei (5.7%) than were found for IVF embryos.     

Recombinant human activin A stimulates development of bovine one-cell embryos matured and fertilized in vitro

The effects of recombinant human activin A on the development of bovine one-cell embryos matured and fertilized in vitro were investigated. In experiment 1, one-cell embryos were cultured in a chemically-defined medium, of modified synthetic oviduct fluid supplemented with 1 mg/ml polyvinyl alcohol (mSOF-PVA), containing different concentrations of activin (0, 0.1, 1, 10, and 100 ng/ml) until 240 hr after in vitro fertilization. The addition of -1 ng/m activin to mSOF-PVA improved development to the blastocyst stage (14.5–17.1%), compared with no addition of activin (5.6%). However, there was no significant difference in hatching rate of embryos among treatments. In experiments 2 and 3, the embryos were also cultured in MSOF-PVA at various periods of exposure to 10 ng/ml activin to evaluate (development to the morula and blastocyst stages, respectively. The proportion of morulae was significantly higher in culture with activin at 20–120 hr postinsemination (37.2%) than with control (25.7%). Total number of cells in morulae at 120 hr postinsemination significantly increased by the addition of activin at 20–72 hr (26.1 cells) and 20–120 hr (24.2 cells) postinsemination, compared with control (20.1 cells). When activin was added to the medium during 20–120 hr and 20–192 hr postinsemination, the percentages of blastocysts (18.0% and 18.7%, respectively) were significantly higher than in the control (9.6%). However, the total number of cells in blastocysts was not significantly different. These results demonstrate that activin stimulates the development of bovine one-cell embryos to the morula and blastocyst stages in vitro. © 1996 Wiley-Liss, Inc.     

Binding of IGF-I to preimplantation rabbit embryos and their coats

It is known that insulin-like growth-factor I (IGF-I) promotes early embryonic development from the morula to the blastocyst stage in rabbits (28). Therefore we used autoradiography to investigate whether IGF-I binds to preimplantation embryos and its coats. From Day 3 after mating onwards, a clear binding of IGF-I to the embryos was observed. There was no difference in binding to the embryoblast or trophoblast cells. Using ligand blot, several IGF-binding proteins (IGFBP; 31 kDa, 33 kDa, 36 kDa, three overlapping bands at 40 to 55 kDa) were obvious in the embryoblast and trophoblast. A 120 to 130 kDa protein was observed exclusively in the embryoblast. Significant binding of (125)I IGF-I to the coats of embryos older than 3 d was detected, and IGF-I was bound via a 38 kDa protein, as detected by ligand blot. To investigate the origin of this protein, the patterns of IGFBP were determined in the oviductal and uterine fluids of pregnant animals (Days 0 to 6). The following binding proteins were observed regularly in the oviductal and uterine flushings: 28 kDa, 32 kDa and 3 overlapping bands in the area of 40 to 55 kDa. In the oviduct the main IGF binding protein was the 32 kDa band (38.7% to 45.9%), while in the uterus it was the 3 overlapping bands at 40 to 55 kDa (42.5% to 24.1%). Because IGF-I is produced in the oviduct and uterus (27), IGFBPs are found in oviductal and uterine fluids, IGF-I is stored in the coats, IGF-I binds to preimplantation embryos and IGF-I promotes early embryonic development (28), the IGF system seams to have a function in the maternal-embryonic interaction.