Hamster Preimplantation Embryos Research Articles

Expression of glucose transporter isoforms and the insulin receptor during hamster preimplantation embryo development

During preimplantation development, embryos of many species are known to express up to five isoforms of the facilitative glucose transporter proteins (GLUT). Development of hamster blastocysts is inhibited by glucose. We therefore investigated GLUT isoform and insulin receptor (IR) expression in hamster preimplantation embryos cultured in glucose-free medium from the 8-cell stage onwards. We show that GLUT1, 3 and 8 mRNA are constitutively expressed from the 8-cell to the blastocyst stage. The IR is expressed from the morula stage onwards. Messenger RNA of the insulin-responsive GLUT4 was not detected at any stage. GLUT1 and 3 were localised by immunocytochemistry. GLUT1 was expressed in both embryoblast and trophoblast, in the latter, mainly in basal and lateral membranes directed towards the blastocoel and embryoblast. GLUT3 was exclusively localised in the apical membrane of trophoblast cells. We show that hamster preimplantation embryos express several GLUT isoforms thus closely resembling embryos of other mammalian species. Despite endogenous IR expression, the insulin-sensitive isoform GLUT4 was not expressed, indicating that the insulin-mediated glucose uptake known from classical insulin target cells may not be relevant for hamster blastocysts.

Embryo-endometrial expression of leukemia inhibitory factor in the golden hamster (Mesocricetus auratus): increased expression during proestrous and window of implantation stages

Leukemia inhibitory factor (LIF) is a pleiotropic IL-6 family cytokine and its maternal uterine expression is critical for mouse blastocyst implantation. In the golden hamster (Mesocricetus auratus), although the blastocyst hatching phenomenon is quite interesting and LIF is shown to regulate hatching, information is not available on the embryonic and uterine expression of LIF and hormonal regulation of LIF expression during the peri-implantation period. The present investigation is aimed at studying embryonic and uterine expression of LIF during preimplantation hamster development. We observed embryonic expression of LIF mRNA and protein in the 8-cell, morula and blastocyst stages. In cycling females, uterine LIF mRNA expression was maximal during the oestrogen-dominant phase of the oestrous cycle, i.e. proestrous stage. Interestingly, during pregnancy, both LIF mRNA and protein were highly upregulated on Days 3.5 and 4 ('window of implantation'), implying a role for this cytokine in blastocyst hatching and implantation. Cell type-specific localisation of LIF mRNA and protein was observed predominantly in luminal epithelium and uterine glands with faint staining being detected in the stroma. The hamster uterus encoded a approximately 4.2 kb LIF transcript whose coding region, when cloned and sequenced, showed a high degree of identity to the murine cDNA counterpart. These data demonstrate that: (1) hamster preimplantation embryos show LIF mRNA and protein expression; (2) uterine expression of LIF mRNA and protein was dependent on elevated levels of circulating oestrogen, and (3) there is a possible functional association of LIF with the peri-implantation development in the golden hamster.

Relationship Between p62 and p56, Two Proteins of the Mammalian Cortical Granule Envelope, and Hyalin, the Major Component of the Echinoderm Hyaline Layer, in Hamsters1

Mammalian cortical granules contain two polypeptides (p62 and p56) that are incorporated into the cortical granule envelope after fertilization and function in cleavage of the zygote and the preimplantation blastomeres. Since the echinoderm hyaline layer and mammalian cortical granule envelope are analogous, and since the hyaline layer protein, hyalin, functions in early echinoderm embryogenesis, this study was done to determine whether p62 and p56 and/or other components of the mammalian cortical granule envelope are related to hyalin. A polyclonal antibody (IL2) against purified S. purpuratus hyalin was shown by confocal scanning laser microscopy to bind to hamster cortical granules and to the cortical granule envelope of fertilized hamster oocytes and preimplantation embryos up to the blastocyst stage. In immunoblots, IL2 bound only to 62- and 56-kDa cortical granule proteins that were incorporated into the cortical granule envelope after fertilization. IL2 binding antigens appeared to be resynthesized by preimplantation embryos starting at the 2-cell stage of development. In vivo treatment of 2-cell-stage hamster embryos with IL2 inhibited blastomere cleavage, but treatment of morulae did not inhibit blastocyst implantation. These results support the idea that the mammalian cortical granule envelope proteins, p62/p56, share a common antigenic epitope(s) with echinoderm hyalin, and that p62/p56, like hyalin, play a role in early embryogenesis.

Regulation of intracellular pH in hamster preimplantation embryos by the sodium hydrogen (Na+/H+) antiporter.

This study was an investigation of the mechanisms for the regulation of intracellular pH (pHi) by hamster preimplantation embryos. The resting pH values of hamster embryos were similar at the 1-cell (7. 19 +/- 0.34), 2-cell (7.21 +/- 0.21), and 8-cell (7.22 +/- 0.41) stages. Cleavage-stage hamster embryos alleviated intracellular acidosis by activity of the Na+/H+ antiporter. The rate of recovery from acidosis was similar for embryos at 1-cell, 2-cell, and 8-cell stages. When Na+/H+ antiporter activity was inhibited by either incubation in Na+-free medium or the presence of an inhibitor, pHi was unable to recover to initial levels. Instead, pHi remained acidic. The Na+/H+ antiporter was also found to contribute to baseline pH regulation, as incubation in Na+-free medium resulted in an immediate intracellular acidification. The set point for Na+/H+ antiporter was pH 7.14. There was no evidence at any developmental stage for activity of either Na+-dependent HCO3-/Cl- exchanger or H+-ATPase in the regulation of pHi. Inhibition of the Na+/H+ antiporter by an amiloride derivative significantly reduced the ability of 2-cell embryos to develop in culture when challenged with acidosis, indicating that the Na+/H+ antiporter is an essential regulator of pHi.

Intracellular divalent cation homeostasis and developmental competence in the hamster preimplantation embryo.

The intracellular magnesium and calcium ion concentrations of in vivo-developed 2-cell hamster embryos were measured using ratiometric fluorometry. Intracellular magnesium and calcium ion concentrations were found to be 0.369 +/- 0.011 mM and 129.3 +/- 7.5 nM respectively. Culture of 1-cell hamster embryos for 24 hr to the 2-cell stage in control medium containing 0.5 mM magnesium and 2.0 mM calcium resulted in approximately a threefold increase to 343.5 +/- 8.0 nM in intracellular calcium ion concentration, while magnesium ion levels were not altered (0.355 +/- 0.007 mM). Increasing medium magnesium concentrations to 2.0 mM significantly increased intracellular magnesium ion concentrations of cultured 2-cell embryos with a concomitant reduction in intracellular calcium ion concentrations. Furthermore, increasing the medium magnesium concentration to 2.0 mM significantly increased development of 1-cell embryos collected at either 3 or 9 hr post-egg activation to the morula/blastocyst and blastocyst stages. Resultant blastocysts had an increased total cell number and increased development of the inner cell mass. Most important, however, culture with 2.0 mM magnesium increased the fetal potential of cultured 1-cells twofold. Therefore, because highest rates of development were observed in a medium that resulted in reduced intracellular calcium ion concentrations, it appears that altered calcium homeostasis is associated with impaired developmental competence of 1-cell embryos in culture.

Glucose and phosphate toxicity in hamster preimplantation embryos involves disruption of cellular organization, including distribution of active mitochondria.

While perinuclear clustering of active mitochondria, as revealed by Rhodamine 123 staining and confocal microscopy, is part of normal hamster embryo development, it is not known whether this reorganization is necessary for development. To determine if disruption of mitochondrial organization occurs in developmentally compromised embryos, the intensity of Rhodamine 123 staining was quantitated using NIH Image Software in different regions of cultured hamster 2-cell embryos exposed to either blocking (contains glucose and phosphate) or non-blocking culture conditions. Three regions within each blastomere were defined based on the organization of freshly collected embryos: cortical (ring beneath plasma membrane), perinuclear, and intermediate regions. While there was no treatment effect on the total staining intensity, glucose and phosphate treated embryos had significantly higher Rhodamine 123 staining in the intermediate region, with corresponding reduced intensity in the perinuclear region, implicating glucose and phosphate in the redistribution of mitochondria. Glucose and phosphate treatment also selectively reduced the FITC Phalloidin staining of actin microfilaments in the interior of the embryo. Neither cytochalasin D nor colchicine, at doses that blocked the second cleavage, caused redistribution of mitochondria like that seen with glucose and phosphate treatment. Additionally, cytochalasin D was unable to disrupt actin microfilaments in the perinuclear region, although it induced a "clumpy" appearance in both mitochondria and microfilaments. This report not only offers a more mechanistic explanation of the embryo 2-cell block (translocation of mitochondria involved in glucose and phosphate inhibition) but suggests that appropriate cellular organization, including the spatial positioning of the mitochondria, may be a prerequisite for normal development and that the physical organization of the embryo is susceptible to damage by exposure to culture conditions.

Succinate and malate improve development of hamster eight-cell embryos in vitro: confirmation of viability by embryo transfer.

The in vitro development of hamster preimplantation embryos is supported by non-glucose energy substrates. To investigate the importance of embryonic metabolism, influence of succinate and malate on the development of hamster 8-cell embryos to blastocysts was examined using a chemically defined protein-free modified hamster embryo culture medium-2 (HECM-2m). There was a dose-dependent influence of succinate on blastocyst development; 0.5 mM succinate was optimal (85.1% +/- 3.9 vs. 54.5% +/- 3.5). In succinate-supplemented HECM-2m, blastocyst development was reduced by omission of lactate (68.5% +/- 7.2), but not pyruvate (85.8% +/- 6.2) or glutamine (84.1% +/- 2.1). Succinate along with either glutamine or lactate or pyruvate poorly supported blastocyst development (28%-58%). Malate also stimulated blastocyst development; 0.01 mM malate was optimal (86.3% +/- 2.8). Supplementation of both succinate and malate to HECM-2m supported maximal (100%) blastocyst development, which was inhibited 4-fold by the addition of glucose/phosphate. The mean cell numbers (MCN) of blastocysts cultured in succinate-supplemented HECM-2m was higher (28.3 +/- 1.1) than it was for those cultured in the absence of glutamine or pyruvate (range 20-24). The MCN was the highest (33.4 +/- 1.6) for blastocysts cultured in succinate-malate-supplemented HECM-2m followed by those in succinate (28.3 +/- 1.1) or malate (24.7 +/- 0.5) supplemented HECM-2m. Embryo transfer experiments showed that 29.8% (+/- 4.5) of transferred blastocysts cultured in succinate-malate-supplemented HECM-2m produced live births, similar (P > 0.1) to the control transfers of freshly recovered 8-cells (33.5% +/- 2.0) or blastocysts (28.9% +/- 3.0). These data show that supplementation of succinate and malate to HECM-2m supports 100% development of hamster 8-cell embryos to high quality viable blastocysts and that non-glucose oxidizable energy substrates are the most preferred components in hamster embryo culture medium.

What is the relationship between the metabolism of preimplantation embryos and their developmental competence?

What is the relationship between the metabolism of preimplantation embryos and their developmental competence?

Translocation of active mitochondria during hamster preimplantation embryo development studied by confocal laser scanning microscopy.

The role of mitochondrial metabolism in the development of preimplantation embryos is unclear. To clarify the importance of mitochondria in early development, the fluorescent probes rhodamine 123 (Rh123: stains active mitochondrial membrane) and nonyl acridine orange (NAO: stains active and inactive mitochondrial membrane) were used with confocal laser scanning microscopy to study the distribution of mitochondria in hamster unfertilized follicular and oviductal eggs (11.5 hr and 16 hr post-hCG, respectively) and preimplantation embryos (1-cell to blastocyst). Rh123 staining indicated that active mitochondria were homogeneously distributed in unfertilized follicular and oviductal eggs. At 3 hr post egg activation (PEA) by sperm, active mitochondria were still found throughout the cytoplasm of the activated egg although they were slightly clustered around the pronuclei and were intensely active in the second polar body. During the next 9 hr, the majority of active mitochondria encircled the apposing pronuclei. By this time the second polar body no longer stained. In 2-cell, 4-cell and 8-cell embryos, there was a striking constancy in the pattern of active mitochondria which were clustered around the nuclei and delineating the cytocortex subjacent to the plasma membrane. In the blastocyst, active mitochondria were most readily detected in the trophectoderm cells in a homogeneous distribution. Staining mitochondria with NAO showed the same distribution patterns as Rh123, indicating that perinuclear clustering of active mitochondria involves the physical movement of these organelles rather than simply changes in their activity. Distribution of actin microfilaments and microtubules showed similar patterns to mitochondria and may be involved in their movement. This migration of mitochondria, beginning during the early stages of fertilization in the hamster egg and persisting until blastocoel formation, must have some functional correlation with successful preimplantation development.

Golden hamster embryonic genome activation occurs at the two-cell stage: correlation with major developmental changes.

The earliest time of onset of embryonic genome activation in golden hamsters was investigated. The inhibition of transcription by alpha-amanitin (11 micrograms/ml) in cultured embryos resulted in a total arrest of development of early 2-cell embryos (26 hr post-egg activation); under similar conditions, immediate cleavage divisions of 1-, late 2-, 4-, and 8-cell embryos were not affected. Electrophoretic analysis of [35S]methionine-labeled embryonic proteins showed that alpha-amanitin treatment apparently inhibited transcription-dependent protein synthesis in early 2-cell and, to some extent, in late 2-cell when compared to 4-cell embryos. Analysis of total RNA synthesis, using [alpha 32P]-UTP or [32P]-orthophosphate, showed that there was a high proportion of radioactivity associated with the macromolecular fraction (RNA) at the early and late 2-cell stages and at the 4-cell stage compared to that at the 1-cell stage. These results indicate that the de novo synthesis of RNA, encoded by the embryonic genome, occurs at the 2-cell stage and that the second and subsequent cleavage divisions of hamster preimplantation embryos are dependent on new transcriptional activity. This initial activity of the embryonic genome in hamsters is coincident with several characteristic features of in vitro development such as a block to development, synthesis of major proteins, change in energy substrate preference, phosphate-inhibition of development and a requirement for amino acids.

Qualitative comparison of protein production at different stages of hamster preimplantation embryo development

The developmental pattern of protein production in hamster preimplantation embryos was investigated as a preliminary to studying the regulation of gene expression. Optimal radiolabelling of embryonic proteins was achieved by culturing a minimum of 40 embryos in 50 μl of Hamster Embryo Culture Medium-2 for 2 h with 10 μCi of freshly lyophilized [ 35S]methionine. Proteins synthesized in vitro by different stages of hamster preimplantation embryos were analysed by one- and two-dimensional polyacrylamide gel electrophoresis followed by autoradiography. There were striking changes in the protein profile following the first cleavage division and also lesser changes after the second cleavage division. There were no detectable qualitative changes in the protein profiles of 4-cell, 8-cell, morula and blastocyst stages although some quantitative difference existed between morula and blastocyst stages. These comparisons of protein profiles during different stages of embryo development indicate that in hamsters the onset of embryonic gene activation occurs during the 2-cell stage.

A protein marker of hamster oocyte fertilization

Using a polyclonal rabbit antiserum directed against guinea-pig heparin-binding placental protein (HBPP), we have demonstrated by indirect immunofluorescence the expression of this antigen by hamster preimplantation embryos. HBPP was localized to blastomeres, but not to the zona pellucida, of hamster preimplantation embryos conceived in vivo. Hamster embryos, from the pronuclear to hatched blastocyst stages, were HBPP-positive with greatest immunofluorescence detected at the pronuclear to 2-cell stages. Thereafter, HBPP-dependent fluorescence diminished. By contrast, mature spermatozoa and unfertilized metaphase II oocytes were HBPP negative. Direct microinjection of spermatozoa or diluent through the zona pellucida and into the ooplasm activated the oocytes to express HBPP. These findings demonstrate that expression of HBPP is a post-conceptional event limited to fertilized (or activated) oocytes and that transcription of paternal genomes was not required for HBPP expression. The distribution and temporal kinetics suggest that HBPP may be secreted or released by activated cortical granules into the perivitelline space and concentrated at the oolemma. Whereas hamster embryos conceived in vivo consistently demonstrated a uniformly distributed immunofluorescence, non-viable and degenerate embryos showed weak and patchy reactions. Thus, we have identified a heparin-interacting protein which may serve as a marker of in-vivo and in-vitro oocyte activation and embryo quality.

Stimulatory and inhibitory effects of amino acids on the development of hamster eight-cell embryos in vitro.

Amino acids were added to a simple medium (TALP) in an effort to enhance the development of hamster eight-cell embryos in vitro. The addition of 1.0 mM glutamine to TALP medium markedly increased the percentage of embryos reaching the blastocyst stage. The addition of groups of 4 or 20 amino acids to TALP + glutamine medium produced differential effects, some groups inhibiting development while others stimulated it. The beneficial effect of glutamine was on the eight-cell to morula transition as well as on blastocyst formation, while the effects of the other 20 amino acids studied were only on blastocyst development. This study represents further progress made in identifying the culture requirements for growth of hamster preimplantation embryos in vitro.

The developmental potential of frozen-thawed hamster preimplantation embryos following embryo transfer: Viability of slowly frozen embryos following slow and rapid thawing

Hamster preimplantation embryos were slowly frozen (0.33°C/min) and seeded above 10°C in TC-199 containing 1.5 M-DMSO. These embryos were thawed either slowly (1.5°C/min) or rapidly (90°C/min). The thawed embryos were examined by morphology, trypan blue exclusion and viability after embryo transfer. Slow thawing gave significantly higher viability compared to rapid thawing. The early preimplantation embryos demonstrate higher sensitivity to freezing. The three tests of viability (morphology, trypan blue exclusion and embryo transfer) were found to be positively correlated.

Metabolism of progesterone by hamster blastocysts and the ontogeny of progesterone metabolic capability.

Progesterone (P) is required for the differentiation of reproductive tracts and maintenance of pregnancy. This study investigates whether the hamster blastocyst is capable of metabolizing P and, if so, at what stage of preimplantation development such capability becomes detectable. When the blastocysts collected from superovulated hamsters on Day 4 of pregnancy were cultured in 0.4 microM P medium, P metabolism was easily detectable at 1.25 h of culture and over half was metabolized by 7.5 h. Two major metabolites were generated: 5 alpha-pregnane-3,20-dione (or 5 alpha-dihydroprogesterone; 5 alpha-DHP) and 5 alpha-pregnane-3 beta-ol-20-one (or allopregnanolone; AP), about 90-95% and 5-10%, respectively. This indicates the activity of two enzymes: delta 4-5 alpha-reductase and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD). The rate of P metabolism increased with P concentration (0.4-6.4 microM), indicating a high capacity of the enzymes. Studies of embryos collected on Days 1-3 showed that P metabolism was not detectable up to 0100 h of Day 3 (2-4-cell), but was detectable with two metabolites, 5 alpha-DHP and AP, at 1515 h of Day 3 (morula) and thereafter. This indicates that, by the morula stage, the hamster embryo has already acquired the enzymatic capability (5 alpha-reductase and 3 beta-HSD) to metabolize P. These results, together with our earlier finding of 17 beta-hydroxysteroid dehydrogenase activity in Days 1-4 embryos, suggest that hamster preimplantation embryos can metabolize both P and estrogens, thus possibly modulating local actions of these hormones and causing local effects in the reproductive tract.

Effect of an estrogen antagonist on development of blastocysts and implantation in the hamster.

CI 628 citrate, an estrogen antagonist, was given as a single intraluminal injection (5 micrograms) on day 3 to ovariectomized, progesterone-treated hamsters. This significantly reduced embryo cleavage rate, transformation of morula into blastocyst, and completely inhibited implantation. The effects of the drug could be reversed by estradiol-17B (1 microgram) but not estradiol-17 alpha (1 microgram) injected intraluminally with CI 628 citrate. Our finding suggests a role of estrogen present in hamster preimplantation embryo in the triggering of embryonic differentiation and implantation of the blastocyst.