Susan Heyner

Preimplantation mouse embryos internalize maternal insulin via receptor-mediated endocytosis: Pattern of uptake and functional correlations

High resolution microscopy in conjunction with colloidal gold-labeled insulin has been used to provide evidence that insulin is internalized by preimplantation mouse embryos by means of receptor-mediated endocytosis and concentration in coated pits. In addition, immunocytochemical analyses at the blastocyst stage, using gold-labeled anti-insulin receptor immunoglobulin (IgG) have confirmed the expression of insulin receptors on all cells of the embryo, including the inner cell mass. Immunocytochemical studies using gold-labeled anti-insulin IgG have provided evidence that the insulin internalized by the embryo is maternally derived. Functional studies show that incubating embryos in physiological levels of insulin results in increased synthesis of RNA and DNA. We conclude that insulin may play a role in early mammalian development, although the precise function of this hormone remains to be defined.

Autoradiographic Evidence for Insulin and Insulin-Like Growth Factor Binding to Early Mouse Embryos

Insulin binding to mouse oocytes and preimplantation embryos was assessed by light-microscopic autoradiography. Significant insulin binding was present on the cells of morulae and increased twofold at the blastocyst stage of development. Insulin binding was markedly decreased by native insulin and to a lesser extent by insulin-like growth factors (IGFs). No specific insulin binding was detected on oocytes or embryos throughout the eight-cell stage. Specific binding of IGF-I and IGF-II was also observed on the cells of blastocyst outgrowths. The findings demonstrate that specific binding of insulin and IGF is temporally expressed on the cells of pre- and peri-implantation mouse embryos. These results confirm and extend our previous immunofluorescence study.

Gene expression in pre-implantation mammalian embryos

The pre-implantation mammalian embryo is initially under the control of maternal informational macromolecules that are accumulated during oogenesis. Subsequently, the genetic program of development becomes dependent upon new transcription derived from activation of the embryonic genome. Several embryonic transcripts including those that encode growth factors, cell junction components and plasma membrane ion transporters are required for normal progression of the embryo to the blastocyst stage. The pattern of genes expressed and the overall program of development is subject to the influences of genomic imprinting as well as external influences encountered by the embryo within the maternal reproductive tract.

Functional foles of insulin and insulinlike growth factors in preimplantation mouse embryo development

SummaryGrowth factors are known to play important roles in cellular proliferation and differentiation. However, little information is available concerning their roles in the earliest stages of mammalian development. The effect of physiologic levels of insulin, insulinlike growth factor-I, and insulinlike growth factor II (IGF-I and-II) on DNA, RNA, and protein synthesis in preimplantation stages of the mouse are described in this study. Quantitative studies of the incorporation of labeled thymidine, uridine, and methionine into trichloroacetic acid-insoluble material by different developmental stages of preimplantation mouse embryos labeled in vitro, indicate that physiologic levels of insulin stimulated DNA, RNA, and protein synthesis with significant effects observed first at the morula stage of development. In contrast, neither IGF-I nor IGF-II stimulated DNA, RNA, or protein synthesis to a significant degree under the same experimental conditions. These results suggest a functional role for insulin at the earliest stages of mammalian embryogenesis.

Sequential analysis of zona thickness during in vitro culture of human zygotes: correlation with embryo quality, age, and implantation.

Zona pellucida thickness was measured daily in zygotes and cleavage stage embryos. Measurements were performed on a Nikon inverted microscope equipped with Hoffman modulation optics, using an ocular micrometer. Zona thickness of each zygote/embryo was measured four times, the zygote/embryo was then “rolled over,” and four more measurements were repeated for a total of eight. The zygotes/embryos were photographed daily and the measurements repeated on the prints. Subsequently, the mean zona thickness for each stage was calculated. A total of 81 patients (mean age 33.8 ± 4.2) participated in the study. A total of 427 embryos were evaluated. Categorical data differences between groups were evaluated by ANOVA and multiple linear regression. For nominal data, the Kruskal‐Wallis test was applied; when P < 0.05 the differences were considered to be significant. We found that the average zona thickness on day 1 of in vitro culture was 17.7 ± 0.14 μm; 16.3 ± 0.14 μm on day 2 and 14.9 ± 0.14 μm on day 3 (P < .0001). When the zona thickness was analyzed in relation to the number of blastomeres on day 3 of culture, there was a highly significant correlation with blastomere number (P < .0001). Similarly, there was a highly significant correlation with embryo grade (P < .005) and fragmentation (P < .001). The data were also analyzed for embryos transferred that resulted in a successful pregnancy, revealing that embryos in a pregnancy cycle had significantly thinner zonae pellucidae (P < .0001), when compared to embryos that were not transferred or from nonconceptual cycles. The average zona thickness also decreased with age, and was most apparent after 35 years. Changes in zona thickness correlated with the number of blastomeres, grade, fragmentation, age and were more evident in embryos transferred from cycles resulting in successful pregnancies. Therefore, zona pellucida measurements should be included in the overall assessment of embryo quality, since this information may be useful in the selection of optimal embryos for transfer. Mol. Reprod. Dev. 47:99–104, 1997.

Stage-specific insulin binding in mouse preimplantation embryos.

Stage-specific insulin binding in the developing mouse embryo was demonstrated by an indirect immunofluorescence technique using an antibody against insulin. Concentration-dependent fluorescence labeling was observed in the morula and blastocyst stages of development, whereas no reactivity was seen in unfertilized oocytes or in 2-, 4-, and 8-cell embryos. The possible significance of these observations is discussed. This represents the first report of stage-specific insulin binding during mammalian preimplantation development.

Analysis of the human zona pellucida during culture: Correlation with diagnosis and the preovulatory hormonal environment

Purpose: The objective of this study was to analyze sequentially the human zona pellucida changes in an in vitro fertilization program as it relates to several variables.Methods: The zona pellucida thickness was measured daily in zygotes and cleavage-stage embryos on a Nikon inverted microscope equipped with Hoffman modulation contrast optics, using an ocular micrometer. A total of 512 embryos from 96 patients was evaluated.Results: There was a highly significant direct correlation between zona thickness and preovulatory estradiol and basal day 3 FSH levels (P < 0.02 andP < 0.0006, respectively). This relationship showed a rapid reversal following 48 hr of culture; embryos from patients with the highest FSH levels had thinner zonae prior to transfer (P < 0.0007). The zonae from patients with unexplained infertility were thicker (19.4 ± 2.7 µm) than those from patients with endometriosis (17.7 ± 2.2 µm), tubal (17.5 ± 2.4 µm), or male-factor infertility (16.4 ± 2.7 µm) (P < 0.0001) on the first day of culture.Conclusions: We hypothesize that the thickness of the human zona pellucida is influenced by the preovulatory hormonal environment and diagnosis. These factors should be considered as part of the embryo quality evaluation prior to transfer or when assessing the possibility of using assisted hatching. More studies are needed to understand the factors regulating the thickness of the human zona pellucida.

Micromachined analytical devices: microchips for semen testing.

Micromachined devices (microchips) have been designed and tested for a range of clinically important assays. In this study we compare sperm motility determined using disposable glass microchips and a conventional Makler chamber. The 17 x 14 mm glass microchips contained three etched test structures each comprising either duplicate or quadruplicate analytical microchannels. Semen samples with sperm counts ranging from 21 to 78 million sperm per ml and forward progression scores of from 1+ to 3+ were evaluated and swimming times ranging from 360 s (3.3+ progression) to 770 s (1+,2 forward progression) observed in the microchips. Motility determined by the time taken for sperm to swim to the end of a microchannel (100 microns wide x 40 microns deep x 10 mm long) in the microchip correlated with forward progression of the sperm determined by the conventional Makler chamber method. This study demonstrates the feasibility of microchips for sperm motility testing and suggests that this technique would be applicable to the study of other types of motile cells.

The role of growth factors in embryo production

Abstract Despite the successful in vitro culture of preimplantation embryos from a number of mammalian species, such embryos are at a developmental disadvantage when compared to their in vivo counterparts. This disadvantage can be overcome to some extent by adding growth factors, or washings from the reproductive tract. It has become clear within the past few years that the oviduct and uterus contain growth factors that can stimulate cellular proliferation, and in some cases, differentiation of preimplantation embryos. These growth factors act along a paracrine pathway by binding to specific receptors on embryonic cells. In addition, the embryo itself produces some autocrine growth factors. The study of these growth factors and their receptors has led to some general conclusions. There are several growth factors that stimulate cellular proliferation, although the precise pathway they utilize is not known. Similarly, it is conceivable there are synergistic interactions of different growth factors required for optimal early development. We summarize information regarding the role of the insulin family of peptides in preimplantation mammalian development and describe some other relevant studies.

Is chromosome 10 a primary chromosomal abnormality in endometrial adenocarcinoma

Seven cases of endometrial adenocarcinoma (EC) are reported. Two of these cases exhibited diploid chromosome ranges and showed simple rearrangements involving a chromosomal abnormality of chromosome 10. In four cases, the chromosome number ranged between 50 and 70; rearrangements were more complex, with many abnormalities such as homogeneously stained regions, minutes, dicentrics, and ring chromosomes. In one case, two subpopulations of cells were detected, one in a diploid chromosome range with chromosome 10 altered, and the second, very pleomorphic. These abnormalities are probably due to the evolution of a destabilized genome and represent a consequence of the advanced stage of the disease. The importance of simple abnormalities as clues to the primary chromosomal change, and the possibility that chromosome 10 represents the primary chromosomal alteration event in EC, are discussed.