Peter L. Kaye

Insulin increases cell numbers and morphological development in mouse pre-implantation embryos in vitro

Insulin, alone or in combination with bovine serum albumin (BSA), was investigated for its effects on cell proliferation and on the proportions of mouse pre-implantation embryos reaching compaction and forming blastocysts during culture in a common basal medium in vitro. Insulin promoted cleavage by 16-20% when added to medium for culture of 2-cell embryos to morulae, blastocysts and expanded blastocysts over 24, 48 and 72 h. These effects on cell division were supported by increases of 65-100% and 31-100% in the rates of compaction and blastocyst formation respectively. The results indicate that the receptor responsible for these actions is probably expressed prior to compaction and possibly at the 4-cell stage. Identical responses to 1.7 and 170 nM insulin suggest that the insulin receptor is capable of mediating both of these developmental effects, although similar mediation by an insulin-like growth factor-1 (IGF-1) receptor is not excluded. BSA, normally a component of culture media, promoted cleavage between 24 and 48 h of culture as well as compaction and blastocyst formation at 15 microM (1 g L-1), probably through nutritional support. Compaction appeared to be promoted by some non-specific action of BSA. Blastocysts that had developed in the presence of both 170 nM insulin and 15 microM BSA contained similar numbers of cells to blastocysts that had developed in vivo.

An Unusual Subcellular Localization of GLUT1 and Link with Metabolism in Oocytes and Preimplantation Mouse Embryos

Abstract Although mouse oocytes and cleavage-stage embryos prefer pyruvate and lactate for metabolic fuels, they do take up and metabolize glucose. Indeed, presentation of glucose during the cleavage stages is required for subsequent blastocyst formation, which normally relies on uptake and metabolism of large amounts of glucose. Expression of the facilitative glucose transporter GLUT1 was examined using immunohistochemistry and Western blotting, and in polyspermic oocytes, metabolism of glucose was measured and compared with that of pyruvate and glutamine. GLUT1 was observed in all oocytes and embryos, and membrane and vesicular staining was present. Additionally, however, in polyspermic oocytes, the most intense staining was in the pronuclei, and this nuclear staining persisted in cleaving normal embryos. Furthermore, GLUT1 expression appeared to be up-regulated both in nuclei and plasma membranes following culture of oocytes in the absence of glucose. In polyspermic oocytes, the metabolism of glucose, but not of pyruvate or glutamine, was directly proportional to the number of pronuclei formed. After compaction, nuclear staining diminished, and GLUT1 localized to basolateral membranes of the outer cells and trophectoderm. In blastocysts, a weak but uniform staining of inner-cell-mass plasma membranes was apparent. The results are discussed in terms of potential roles for GLUT1 in pronuclei of oocytes and zygotes, nuclei of cleavage-stage embryos, and a transepithelial transport function for GLUT1, probably coupled with GLUT3, in compacted embryos and blastocysts.

IGF-2 stimulates growth and metabolism of early mouse embryos

Recent reports indicate that the insulin gene family plays a significant role in early development. Both insulin and IGF-1 stimulate growth and metabolism in preimplantation mouse embryos, however, little is known of the physiological effects of IGF-2. In this study, addition of IGF-2 to defined culture medium for the culture of 2-cell embryos stimulated blastocyst formation by 15%, ICM mitogenesis by 37%, and protein synthesis by 35%. EC50s of 12-63 pM IGF-2 for these responses were in the range for mediation by IGF-2 receptors. These results coupled with the previously demonstrated presence and expression of the IGF-2 receptor from the 2-cell stage supports a role for this third member of the insulin gene family in early development.

The role of growth factors in preimplantation development

It has become clear that the mammalian embryo participates in a complex dialogue with the maternal physiology. The language of the dialogue is growth factor signalling. The embryo expresses receptors for insulin, IGFs, GH, EGF and cytokines including LIF, and CSFs; whilst ligands are secreted by the supporting tissues of the oviduct and uterus, and in some cases, the embryo itself. In the preimplantation period when the embryo is travelling to the uterus and passing through its first differentiation, these ligands affect embryonic physiology, apparently in ways that optimise developmental potential and synchronise embryonic and maternal physiologies. It is not yet clear in most cases whether this is by autocrine, paracrine or endocrine mode. In the crucial peri-implantation phase the embryo is preparing to invade the maternal system for which extensive uterine remodelling is necessary. A model is proposed in which a cascade of growth factor activities, orchestrated by the ovarian steroid patterns, choreographs the biochemical players (ECM proteinases and their inhibitors) which initiate this activity.

FAM deubiquitylating enzyme is essential for preimplantation mouse embryo development

FAM is a developmentally regulated substrate-specific deubiquitylating enzyme. It binds the cell adhesion and signalling molecules beta-catenin and AF-6 in vitro, and stabilises both in mammalian cell culture. To determine if FAM is required at the earliest stages of mouse development we examined its expression and function in preimplantation mouse embryos. FAM is expressed at all stages of preimplantation development from ovulation to implantation. Exposure of two-cell embryos to FAM-specific antisense, but not sense, oligodeoxynucleotides resulted in depletion of the FAM protein and failure of the embryos to develop to blastocysts. Loss of FAM had two physiological effects, namely, a decrease in cleavage rate and an inhibition of cell adhesive events. Depletion of FAM protein was mirrored by a loss of beta-catenin such that very little of either protein remained following 72h culture. The residual beta-catenin was localised to sites of cell-cell contact suggesting that the cytoplasmic pool of beta-catenin is stabilised by FAM. Although AF-6 levels initially decreased they returned to normal. However, the nascent protein was mislocalised at the apical surface of blastomeres. Therefore FAM is required for preimplantation mouse embryo development and regulates beta-catenin and AF-6 in vivo.

Toxic Effects of Hyperglycemia Are Mediated by the Hexosamine Signaling Pathway and O-Linked Glycosylation in Early Mouse Embryos

Maternal hyperglycemia is believed to be the metabolic derangement associated with both early pregnancy loss and congenital malformations in a diabetic pregnancy. Using an in vitro model of embryo exposure to hyperglycemia, this study questioned if increased flux through the hexosamine signaling pathway (HSP), which results in increased embryonic O-linked glycosylation (O-GlcNAcylation), underlies the glucotoxic effects of hyperglycemia during early embryogenesis. Mouse zygotes were randomly allocated to culture treatment groups that included no glucose (no flux through HSP), hyperglycemia (27 mM glucose, excess flux), 0.2 mM glucosamine (GlcN) in the absence of glucose (HSP flux alone), and O-GlcNAcylation levels monitored immunohistochemically. The impact of HSP manipulation on the first differentiation in development, blastocyst formation, was assessed, as were apoptosis and cell number in individual embryos. The enzymes regulating O-GlcNAcylation, and therefore hexosamine signaling, are the beta-linked-O-GlcNAc transferase (OGT) and an O-GlcNAc-selective beta-N-acetylglucosaminidase (O-GlcNAcase). Inhibition of these enzymes has a negative impact on blastocyst formation, demonstrating the importance of this signaling system to developmental potential. The ability of the OGT inhibitor benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside (BADGP) to reverse the glucotoxic effects of hyperglycemia on these parameters was also sought. Excess HSP flux arising from a hyperglycemic environment or glucosamine supplementation reduced cell proliferation and blastocyst formation, confirming the criticality of this signaling pathway during early embryogenesis. Inhibition of OGT using BADGP blocked the negative impact of hyperglycemia on blastocyst formation, cell number, and apoptosis. Our results suggest that dysregulation of HSP and O-GlcNAcylation is the mechanism by which the embryotoxic effects of hyperglycemia are manifested during preimplantation development.

Visualization of insulin receptors on mouse pre-embryos

Because insulin stimulates pre-embryonic protein metabolism and growth, the presence of insulin receptors on early mouse embryos was investigated immunohistochemically, using a specific anti-insulin receptor IgG. Staining was not present on fertilized eggs or on 2-cell, 4-cell or uncompacted 8-cell embryos, but insulin receptors were visible on compacting 8-cell embryos and on morulae and blastocysts. This ontogeny correlates with functional studies showing that insulin affects protein synthesis during these post-compaction stages. Insulin receptors were also present on isolated inner cell masses, which have also been shown to be responsive to insulin. Because the ontogeny of the appearance of insulin receptors and the presence of these receptors on both cell populations in the blastocyst coincide with the stimulatory effects of insulin observed in previously reported functional studies on pre-embryos, we believe that these insulin receptors mediate insulins regulatory actions during early mouse embryogenesis.

Nutrient Sensing by the Early Mouse Embryo: Hexosamine Biosynthesis and Glucose Signaling During Preimplantation Development

Abstract Although mouse oocytes and cleavage-stage embryos are unable to utilize glucose as a metabolic fuel, they have a specific requirement for a short exposure to glucose prior to compaction. The reason for this requirement has been unclear. In this study we confirm that cleavage-stage exposure to glucose is required for blastocyst formation and show that the absence of glucose between 18–64 h after hCG causes an irreversible decrease in cellular proliferation and an increase in apoptosis. More importantly, this glucose signals to activate expression of Slc2a3 transcript and SLC2A3 protein, a facilitative glucose transporter (previously known as GLUT3) associated with developmental competence and increased glucose uptake used to fuel blastocyst formation. Glucosamine could substitute for glucose in these roles, suggesting that hexosamine biosynthesis may be a nutrient-sensing mechanism involved in metabolic differentiation. Inhibition of the rate-limiting enzyme in this pathway, glutamine-fructose-6-phosphate amidotransferase (GFPT), inhibited expression of the SLC2A3 transporter protein and blastocyst formation. Glucosamine, a substrate that enters this pathway downstream of GFPT, was able to overcome this inhibition and support SLC2A3 expression. These data suggest that early embryos rely on hexosamine biosynthesis as a glucose-sensing pathway to initiate metabolic differentiation.

Stimulation of protein synthesis and expansion of pig blastocysts by insulin in vitro

Present evidence indicates that insulin may act as a growth factor during preimplantation development. This hypothesis has been tested on pig blastocysts by determining the effect of insulin on protein synthesis and blastocyst expansion over 24 h. Blastocysts were collected from superovulated gilts or sows on Day 5 or 6 and incubated overnight in a modified BMOC2 medium. Those that were cultured with 1.7 nM insulin had 14% larger radii, and were 36% more active in their incorporation of [3H]leucine (protein synthesis) than those that had been cultured in non-supplemented medium. There was a significant linear correlation between the rate of protein synthesis and the radius of blastocysts when all blastocysts and only those cultured with insulin were examined, but the correlation for the blastocysts in non-supplemented medium was just outside statistical significance. The regression coefficient for the insulin-treated blastocysts was 132% of that for blastocysts cultured in unsupplemented medium; this suggests that insulin increased the size of blastocysts and the rate of protein synthesis per unit size. The results indicate that pig blastocysts respond to physiological levels of insulin in similar fashion to those of mice and cattle, supporting the hypothesis that insulin may act as a general embryonic growth factor. Because of the cross reaction between the insulin receptor and the ligands, insulin and insulin-like growth factor 1 (IGF-1), the results also suggest that IGF-1, reported to be present in pig uterine fluid, could be involved in this stimulation in utero.

The role of insulin-like growth factor II and its receptor in mouse preimplantation development

Insulin-like growth factor II (IGF-II) and its receptor, the IGF-II/mannose-6-phosphate (IGF-II/M6P) receptor, are first expressed from the zygotic genome at the two-cell stage of mouse development. However, their role is not clearly defined. Insulin-like growth factor II is believed to mediate growth through the heterologous type 1 IGF and insulin receptors, whereas the IGF-II/M6P receptor is believed to act as a negative regulator of somatic growth by limiting the availability of excess levels of IGF-II. These studies demonstrate that IGF-II does have a role in growth regulation in the early embryo through the IGF-II/M6P receptor. Insulin-like growth factor II stimulated cleavage rate in two-cell embryos in vitro. Moreover, this receptor is required for the glycaemic response of two-cell embryos to IGF-II and for normal progression of early embryos to the blastocyst stage. Improved development of embryos in crowded culture supports the concept of an endogenous embryonic paracrine activity that enhances cell proliferation. These responses indicate that the IGF-II/M6P receptor is functional and likely to participate in such a regulatory circuit. The functional role of IGF-II and its receptor is discussed with reference to regulation of early development.