The results depicted below derive from the pooled results from four repetitive experiments. The cleavage and blastocyst rates of the embryos were investigated daily until day 4.3 post-fertilization using an inverted microscope. More embryos developed into the blastocyst stage for the IGFBP-1/IGF-1 group than was the case for the control group (94.6% vs. 81.9% respectively; p = 0.05). The hatching rate for the IGFBP-1/IGF-1 group was significantly higher than the corresponding figure for the control group (68.8% vs. 48.6%; p = 0.026; Table 3).

The results shown below are pooled from three repeats of the culture experiments. The developmental stages for the cultured embryo were determined daily until day 4.1 post-fertilization using an inverted microscope. They were evaluated before hatching on the morning of day five, that is day 4.1 post-fertilization, instead of day 4.3 post-fertilization, because the hatching of the cultured embryos may distort the integrity of their figures and therefore interfere with the counting of the total cell number. In order to enumerate the total cell number of cultured embryos, the embryos that progressed to the blastocyst stage for both groups were cultured in Hoechst H33342 medium (5 μg /ml) for three to five minutes, and then mounted onto a slide and examined under a fluorescence microscope. The results of cell enumeration are shown in Table 4. There appeared to be a significantly-increased total cell count for the blastocysts deriving from the IGFBP-1/IGF-1-supplemented group than was the case for the control group (87.6 ± 5.3 vs. 73.7 ± 7.1 respectively, p < 0.01; Figure 3).

Zygotes recovered from female mice (C57BL/6 J x C3H), naturally mated with C3H males, were cultured in kSOM + amino acids (kSOM/AA) medium alone, and served as the control group. The culture medium used for one experimental group was supplemented with IGF-1 (100 ng/ml), whilst the medium for the other experimental group was supplemented with an IGFBP-1/IGF-1 complex (100 ng/ml). The randomly-selected hatching blastocysts deriving from these experimental groups were placed into feeder wells for subsequent establishment of the stem-cell lines used herein. The embryonic stem-cell lines were established from the inner-cell mass (ICM) of the hatched blastocyts following 23-25 days of co-culture with an STO feeder-cell layer, such a procedure resulting in an increased proportion (percentage) of successfully-established stem-cell lines (45% vs. 59.6% vs. 27.3% respectively, p < 0.05; Figure 4).

Autocrine and paracrine secretion of growth factors, and the timely expression of relevant specific receptors would likely affect the success of in vitro culture of the embryos 71727. The deprivation of necessary growth factors for such embryos has been shown to trigger apoptosis for a wide variety of in vitro cultured cells 4. In recent studies pertaining to apoptosis, it has been suggested that TGF-α plays a critical role in the regulation of apoptosis as a "survival factor" for mouse blastocysts in vitro.3. Other putative survival factors for in vitro cultured embryos include IGF-1 and PAF (platelet activating factor) 41928. It has been previously demonstrated that culture-medium supplementation with IGF-1 promoted the development of bovine embryos to the morula stage at day five, and that the addition of 10 μg/ml insulin to the medium increased the cell number of bovine blastocysts cultured in vitro 28. Significantly-enhanced effects were shown for LIF, IL-6, TGF-α, EGF, IGF-1, IGF-2, and TGF-βsupplement as regards improving embryonic hatching of post-thawed mouse morulae, in particular, the hatching rate, the level of which ranged from 61% for the control group to 82% for the IGF-1-supplemented group 14.

It has previously been suggested that the core anti-apoptotic effect of IGF-1 was mediated via IGF-1R 122930. Further, the IGF-1 receptor can be activated by either IGF-1 or insulin, however, the relative dimension of the anti-apoptotic effect elicited by IGF-1 upon the IGF-1 receptor would appear to be more efficient than is the case for insulin 31. Although the IGF-1 receptor and the insulin receptor utilize a common pathway through IRS-1 (Insulin receptor substrate-1) and PI3-kinase (phosphatidylinositol 3-kinase), the IGF-1 receptor can act through an alternative anti-apoptotic pathway which appears to be IRS-1 and PI3-kinase-independent and is not shared with the insulin receptor 32. This suggests that the mitogenic activity of IGF-1 through the IGF-1 receptor, which correlates with the tyrosine kinase domain, may be able to elicit a more-powerful anti-apoptotic effect than expected anti-apoptotic effect through pathway of the IRS-1. Therefore, further studies are needed.

In our study, the benefit of supplementation of culture medium with IGF-1 for the development of in vivo fertilized embryos has clearly been demonstrated (Table 1). The blastocyst formation rate was significantly increased for the IGF-1-supplemented group as compared to the control group (88.7% vs. 81.9% respectively), although the significance did not appear to be particularly prominent or dramatic (p = 0.043). This may be due to the moderately high blastocyst-formation rate (81.9%) for our control group, in which the apoptotic factors might be less than those in other in vitro studies with low blast-formation rate.38222533. In the studies of Lighten et al. (1998), treating cultured human embryos with 1.7 nM IGF-1 increased the rate of blastocyst formation by 25%, (60% versus 35% for the control; p= 0.0018).25. There were low blastocyst-formation rate in their control group (35%), in which suboptimal conditions with apoptotic factors may be encountered. The relative increase in the level of blastocyst formation was greater and more dramatic in their study as compared to ours.25. The anti-apoptotic effect of IGF-1 noted here would appear to be mainly due to its role as a protector of embryonic cells from possible injury 112034. Therefore, as regards elevating blastocyst formation rate, the more-prominent effect of IGF-1 would likely be determined under the more suboptimal culture conditions. Herrler and his colleagues (1998) have demonstrated that IGF-1 might act as a "survival factor" during preimplantation development, in which situation the apoptosis was induced by UV irradiation (254 nm wave length; 11.8 W/m²).22. Indeed, our studies have demonstrated that the supplementation of culture medium by IGF-1 would likely improve the blastocyst-formation rate of the in vivo fertilized embryo cultured in vitro. This significant but not prominent effect of IGF-1, when compared to the results obtained in other studies with imposed apoptotic factors 1422, might have resulted from the absence of the induction of a significant level of apoptosis in our experimental study.

To study the survival effect of IGF-1 alone over the kSOM cultured embryos, we observed the necrotic and apoptotic changes as revealed by Annexin V and PI staining using an epifluorescent microscope. As indicated in Table 2, a significantly-lower number of apoptotic and necrotic embryos were observed for the IGF-1-supplemented group than was the case for the control group (9.4% vs. 24.1% respectively, p < 0.05). Further, we also noted that there were fewer Annexin V-revealed apoptotic cells amongst blastocysts for the IGF-1 group as compared to the control group (n= 0 vs. n = 5 respectively). Such results reveal a significant anti-apoptotic and, subsequently, an anti-necrotic effect elicited by IGF-1 supplementation of embryo culture medium.

The proportion (percentage) of apoptotic blastocysts, revealed by only Annexin V fluorescence staining at the termination of embryo culture in kSOM/AA medium either with or without IGF-1 supplementation was 3.1% (5/162), a result that appeared to be quite different from that of Kamjoo and his colleagues in 2002 6, from whose studies, the apoptosis index (dead cells/total cells) revealed with TUNEL staining for mouse embryos cultured in kSOM was 14.4 % 1. We were unable to determine the dead-cell count for each blastocyst, because the quality of the Annexin V fluorescence in the blastocysts appeared to be quite scant in our study, although the total cell number of the blastocysts examined in our study was higher than that reported by Kamjoo and his colleagues in 2002 6.

In our study, the embryos were collected from F1 female C57BL/6 mice mated with C3H males and were cultured in kSOM/AA culture medium. Such a model appears to demonstrate a better potential to promote blastocyst formation in culture and to result in an increased blastocyst total cell number than the models with other culture media and strain of mouse 635. The blastocyst formation rate for the control group in our study was 81.9%. By using a MF1 x MF1 strain and C57BL6/CBA x C57BL6/CBA mice, Kamjoo and his colleagues (2002) were able to report a blastocyst formation rate of 62% (total cell number = 78.0, apoptotic index = 20.3%) and 98% (total cell number = 55.0, apoptotic index = 14.4%), respectively for these two strains 6. Therefore, blastocysts deriving from MF1x MF1 revealed a higher apoptotic index than did those deriving from C57BL6/CBA x C57BL6/CBA (20.3% vs. 14.4% respectively) cultured in kSOM medium 6. Such difference in results may be attributable to the different types of mouse strain used for the different experiments 6.

Quite independently of the type of mouse strain used for experimentation, different methods for the determination of apoptosis, such as Annexin V, TUNEL, 4'-6-diamidino-2-phenylindole, in situ nick translation, and Bcl-2 and Bax protein levels, may also make for different results of detection of apoptosis.68132236. In the studies of Berlerkom and Patrick in 1998 pertaining to apoptosis with TUNEL and Annexin V staining over the oocytes, most Annexin V fluorescence appears subsequent to the documented fragmentation of the oocytes, although such an event appeared to occur coincidentally with PI staining.36. Annexin V fluorescence, however, was reported by Berlerkom and Patrick to have only appeared to be associated with positive PI fluorescence, thus there were no oocytes that were stained with only Annexin V and not featuring PI fluorescence in their studies.36. In these workers' studies, no temporal or spatial relationship between the appearance of TUNEL- and annexin V-stained fragments in the same oocyte was reported.36. In our embryo studies, the process of apoptosis for embryos did not proceed by way of the oocytes; the plasma membrane alternations with the associated translocation of phosphatidyserine from the inner layer to the outer layer, which was revealed by Annexin-V FITC staining, occurred prior to (Figure 2A) or coincidentally with the appearance of PI-stained necrotic changes (Figure 2B). Further, we noted that 48.1% (13/27) of arrested embryos exhibited the Annexin V stain only (Figure 2A), although 44.4% (12/27) of arrested embryos did exhibit PI-stained neuclei, for which the Annexin V stain was frequently observed coincidentally (Figure 2B). This arose because Annexin V was also able to stain the inner plasma membrane of ruptured necrotic cells.

Early in our studies, we had tried some preliminary investigation with TUNEL evaluation for apoptosis at the completion of embryo culture. The frequency of observed TUNEL fluorescence for blastocysts in vitro appeared to be higher than that of the Annexin V fluorescence, the latter appearing to be extremely rare for the well-developed blastocysts. Therefore, the observed apoptotic changes involving the DNA fragmentation of blastomeres would appear to have resulted from the activation of an endogenous nuclear endonuclease, which was labeled by the fluorescein-dUTP in the TUNEL staining technique.37. Such a change appeared to occur more frequently and earlier for blastocysts than did the phosphatidylserine translocation in the plasma membrane during the apoptotic changes of blastocysts. Therefore, in the study of Kamjoo et al. (2002), apoptotic blastomeres were frequently seen, this study revealing an apoptotic index of 14.4% for the embryos collected from C57BL6/CBA x C57BL6/CBA mice 6. In our study, 8.5–15.6% of the cultured embryos exhibited only Annexin V stain positivity, in which cases the necrotic embryos were excluded by the PI stain. If this coincidental PI-revealed necrosis in Annexin V stained embryo was not excluded from the statistic counting of apoptotic changes of the cultured embryos, there would be a total of 9.4–24.1% of Annexin V-stained embryos exhibiting ongoing apoptotic changes. Since there is no temporal or spatial relationship between the occurrence of TUNEL- and annexin V-staining positivity for the degenerating oocytes, these relationships during embryo development still need to be investigated further.36.

As shown, we had demonstrated that IGF-1 decreased the level of apoptosis of mouse embryos in vitro. The question thus arises, how is IGF-1 regulated and transported in the microenvironment during the preimplantation period, and, further, will IGFBP-1 decrease or increase the influence of IGF-1? Following on, a further question arises will IGF-1 be transported into the ICM of an embryo? The variation in apoptosis with different microenvironments and different strains of test animals is likely to be the result of variable embryonic expression of intracellular and extracellular regulators. Herrler et al. (1998) used UV radiation as an apoptotic factor to demonstrate that IGF-1 acted principally and significantly as a " survival factor".22.

IGFBP-1 is a 25 kD protein produced predominantly by hepatocytes and decidualized ovarian endometrium 1620. IGFBP-1 provides a transport vehicle for IGF-1 and IGF-II, and controls the distribution of IGF-1 and possibly directs it to a specific receptor site 1620. IGFBP-1 also controls IGF action at the cellular level by either restricting or augmenting the access of IGF-1 to an appropriate receptor 1620. Further, IGFBP-1 appears to reveal a very high affinity for IGF-I and IGF-II 1620, therefore, it usually sequesters IGF-I, acting as a reservoir and inhibitor of IGF-1 by preventing its binding to the IGF-I receptor site 20.

IGFBP-1 may demonstrate both stimulatory and inhibitory effects upon IGF-1-induced DNA synthesis 20. These effects relate, predominantly, to the degree of phosphorylation and de-phosphorylation of the IGFBP-1 molecular species, and three mechanisms, including phosphorylation, proteolysis, and the association with the extracellular matrix (ECM)/integrin, influence the dissociation of IGF-1 from IGFBP-1 1620. Phosphorylation of the molecule increases the affinity of IGFBP-1 for IGF-1 and inhibits IGF-1 binding to its receptor, such that de-phosphorylation of IGFBP-1 reduces its affinity for IGF-1 resulting in increased IGF-1 bioavailability 1620. Therefore, the ratio of non-phosphorylated or a lesser phosphorylated form to the phosphorylated form of IGFBP-1 determines the level of IGF-1 bioactivity in vivo 2038. IGFBP-1 can be effectively proteolysed into fragments, which display a reduced affinity for IGF-1, such that following phosphorylation of IGFBP-1, IGF-1 will be released in a free form to bind with the IGF-1 receptors subsequent to the proteolysis step 2139. The association between IGFBP-1 and ECM/integrin results in a decreased affinity for IGF-1, thus the IGFBP-1/IGF-1 complex is modulated by ECM/integrin interaction 1620213940. As for the extracellular regulators, the role of IGFBP-1 deserves mention in this regard here, as does further study of that role since little free IGF is detectable in the human serum in vivo. Most IGFs are bound to larger proteins and only as little as 0.4% of total IGF-1 is unbound and free in the serum 41.

As a coordinating factor, we chose the dephosphorylated IGFBP-1 molecular complex to evaluate the influence of IGFBP-1/IGF-1 upon embryo development, IGFBP-1 being clearly vital to the expression of IGF-1. In the results of our experiments as depicted in Table 3, the synergistic effect of the IGFBP-1/IGF1 complex embryo development was significant. The rates of blastocyst formation and hatching were both significantly elevated for the experimental groups of IGFBP-1/IGF-1 as compared to the control group (blastocyst formation: 94.6% vs. 81.9%, p = 0.05; hatching: 68.8% vs. 48.6%, p < 0.05). The IGFBP-1 component of the IGFBP-1/IGF-1 complex used in our studies was initially isolated from the human hepatoma cell line Hep G2, and was found to exist primarily in its phosphorylated form. To obtain the IGFBP-1/IGF-1 complex, the IGFBP-1 moiety was dephosphorylated with alkaline phosphatase and reacted with IGF-1 in a 1:1 ratio (as per product information in Sigma No 12024; Sigma catalogue). Therefore, it seems likely that the "used" IGFBP-1 in our studies should be all dephosphorylated and demonstrate a low affinity for IGF-1, such that the resultant sequestration of IGF-1 due to the binding of IGF-1 to IGFBP-1 should be minimal. Further, the IGF-1 molecule was able to be deactivated in the culture medium in the presence of fetal calf serum (FCS), and the dephosphorylated IGFBP-1, in the form of an IGF-1/IGFBP-1 complex, was able to play a role in the preservation and carriage of IGF-1. This effect appears to be more important during the peri-implantation period and later embryo development than at earlier stages of embryo development 42.

Several growth factors have been reported to be anti-apoptotic; LIF, IL-6, TGF-α, EGF, IGF-1, IGF-II, and TGF-β etc., all appearing to be beneficial to post-thawed mouse blastocysts as regards expansion in culture and subsequent hatching 3. LIF has been proposed to play a role in maintaining the proliferative ability of ICM cells, yet it failed to increase the total cell number of cultured blastocysts, whilst TGF-α has been reported to demonstrate a marginal potential to increase the cell number of blastocysts 3. There would appear to be several different pathways for these growth factors to benefit the embryos to which they are exposed in culture, and also, there appear to be several parameters that can be assessed in order to reveal the relative positive effects of such growth factors, these including cell number and accelerated development, embryo grading, hatching ratio, and even zona pellucida thickness variation 43. The ultimate outcome for the blastocyst is to become a viable embryo featuring an abundance of pluripotential stem cells before and shortly after implantation 44. Therefore we choose cell number per blastocyst and the passage of established stem-cell culture as the landmarks of the beneficial effect of growth factors upon embryo development for such potent mitogenic agents as IGF-1 14.

For the evaluation of the effects of the IGF-1/IGFBP-1 complex upon the total cell count of the blastocysts, we randomly choose a selection of embryos cultured in medium supplemented with IGF-1/IGFBP-1 complex and a non-supplemented analogue for Hoechst 33342 staining. Fifteen embryos deriving from the IGFBP-1/IGF-1 group and 18 embryos from the control group were able to be scored after having been successfully stained with Hoechst 33342. This BisBenzimide fluorescent DNA stain is plasma membrane permeable, and intercalates in the A-T regions of the DNA molecule, it thus being useful for staining DNA, chromosomes, and nuclei 45. Thus, Hoechst 33342 is able to stain the nuclei of both viable and non-viable cells. Therefore, the total cell count of the blastocysts which were enumerated for cell number was determined by counting the total number of stained nuclei at the end of culture using fluorescent microscopy. Use of the Hoechst 33342 stain revealed that there was a significant increase in the total cell number of blastocysts in the IGFBP-1/IGF-1 group than was the case for the control group (87.6 ± 5.3 vs. 73.7 ± 7.1 respectively).

The total cell number of the blastocyst is dependent upon the developmental environment and the particular stage of the embryo at which it is assayed 644. Over a similar time course, blastocysts that developed in vivo revealed a higher cell number than those cultured in vitro 6. From the results of Kamjoo's group (2002), the total cell number of the murine blastocyst flushed from the uterus on day four was, respectively, 87.4 ± 2 and 61.3 ± 4.4 cells for mice of the C57BL6/CBA x C57BL6/CBA and MF1 x MF1 strains of mice 6. In the same study, the average day-four TCN of blastocysts developed from in vivo fertilized embryos cultured in vitro with kSOM was 55.0 ± 2.3 and 66.9 ± 1.6 cells for, respectively, C57BL6/CBA x C57BL6/CBA and 57BL6/CBA x MF1 mouse strains 6. In our study, the embryos were flushed out from the oviducts on day zero, after the confirmation of a vaginal plug having developed. The in vivo fertilized embryos were then cultured in kSOM/AA-supplemented medium either in the presence or absence of IGFBP-1/IGF-1 until day 4.1 post-fertilization (day five morning). For our control group (with no IGFBP-1/IGF-1 supplementation), the mean TCN of the blastocysts was 73.7 ± 7.1 (Table 4), such a figure being similar to that for the MF1 x MF1 embryo in kSOM-supplemented culture on the morning of day five following pregnancy, as has been reported previously 6.

Certain apoptotic factors have been noted to be present in the in vitro culture of mouse embryos, as evidenced by the observation of the occurrence of developmental retardation and the level of apoptosis having both increased amongst mouse embryos following in vitro culture 646. The embryos cultured in vitro which is clearly a suboptimal condition as compared to the natural environment in the uterus, have not only been deprived of a whole family of necessary growth factors but have also faced exposure to a variety of apoptotic factors in the in vitro environment, suggesting that a decrease in total cell number of the blastocyst would likely be expected.

Early differentiation of the embryo leads the development of two distinct lineages-the inner cell mass and the trophectoderm 4447. For the mouse, a number of immortal stem-cell lines can be obtained from both cell types 48. The pluripotent internal cell component of the blastocyst, the ICM, is segregated into a subcompartment, the epiblast, prior to blastocyst implantation 444. It is evident that ES cells originate from the epiblast, and isolation of epiblast cells has been reported to enhance the efficiency of ES cell derivation 49. Prior to the induction of diaphase (implantation delay), an increase in epiblast cell numbers appears to enhance the efficiency of ES cell generation 505152. In our study, the significantly-increased success in the establishment of a stem-cell line as compared to control group was the indirect evidence suggesting an increased level of viable cells in culture and a decreased level of apoptosis for the blastocysts cultured in medium supplemented with IGF-1 and IGFBP-1/IGF-1.

All embryos used in this study were collected from F1 female mice (C57BL/6 J x C3H, six to eight weeks old) mated with C3H males. Mice were maintained under a daily light regime of 14 hours L: 10 hours D (Light: Dark). Female mice were superovulated by an injection of pregnant mare serum gonadotropin (5IU; PMSG, Sigma, St Louis, MO, USA) at 5 p.m., followed by an injection of human chorionic gonadotrophin (5IU; hCG, Sigma, St Louis, MO, USA) 48 hours later. Females were placed in cages with males subsequent to the administration of hCG to the former. On the following morning, the females were examined for the presence of a vaginal plug. Females exhibiting a vaginal plug were sacrificed 16 hours post-hCG injection by means of cervical dislocation and their oviducts were excised. The in vivo fertilized zygotes were then flushed out from the oviducts of these mice 47. The embryos so collected were randomly allocated to control and treatment groups with different in vitro culture conditions. In each repeat experiment, three to four pairs of successfully mated mice were used and the results of each experiment were pooled from several repeats.

The culture medium used in our study was kSOM/AA medium 23243555 and all other chemicals used in this study were purchased from Sigma Chemicals unless otherwise specified.