Different Stages Of Preimplantation Development Research Articles

Understanding Chromosome Structure During Early Mouse Development by a Single-Cell Hi-C Analysis.

Over the past two decades, the development of chromosome conformation capture technologies has allowed to intensively probe the properties of genome folding in various cell types. High-throughput versions of these C-based assays (named Hi-C) have released the mapping of 3D chromosome folding for the entire genomes. Applied to mammalian preimplantation embryos, it has revealed a unique chromosome organization after fertilization when a new individual is being formed. However, the questions of whether specific structures could arise depending on their parental origins or of their transcriptional status remain open. Our method chapter is dedicated to the technical description on how applying scHi-C to mouse embryos at different stages of preimplantation development. This approach capitalized with the limited amount of material available at these developmental stages. It also provides new research avenues, such as the study of mutant embryos for further functional studies.

Expression of enzymes involved in the synthesis of prostaglandin E2 in early- and late-cleaved bovine embryos at different stages of preimplantation development

Prostaglandin (PG) E2 plays a role in numerous aspects of mammalian reproduction, such as oviductal transport of gametes, hatching from the zona pellucida in blastocysts and early embryonic development. Despite the evident role of PGE2 in the regulation of female reproductive processes, in the literature, there is very little information concerning the expression of PGE2 synthesizing enzymes and the exact amount of PGE2 produced by bovine embryos in vitro. In the present study, we aimed to determine the mRNA levels and immunolocalization of the enzymes responsible for PGE2 synthesis (PTGS2, mPGES1, mPGES2 and cPGES) in embryos at the 2-cell, 4-cell, 8-cell, 16-cell, morula, early blastocyst, blastocyst, expanded blastocyst and hatched blastocyst stages, using a well-defined bovine model of oocyte developmental competence based on the time of first cleavage. PTGS2, mPGES2 and cPGES transcripts and proteins were detected in all stages of embryos, whereas the mPGES1 transcript and protein were not detected in embryos from the 2- to 16-cell stage. The results showed different transcription profiles of the enzymes involved in PGE2 synthesis in early- and late-cleaved embryos during the early stages of their in vitro preimplantation development. We also found that all the analysed stages of bovine preimplantation embryos released PGE2, with the highest concentration on Day 7 of culture in both the early- and late-cleaved groups. The present study is the first to demonstrate PGE2 synthesis and production by bovine early- and late-cleaved embryos at different stages of preimplantation development. Bovine embryos can produce PGE2, which may exert paracrine regulation during development. The transcription levels of PGE2 synthases were affected by the embryonic stage of development and quality. Our results indicate that the different transcription profiles of PTGS2, mPGES1, mPGES2 and cPGES, as well as PGE2 concentration, in early-versus late-cleaved embryos are dependent on the quality of the oocytes from which the embryos were obtained, which could reveal the association of PGE2 production during bovine preimplantation development with more advanced stages of embryo development.

2 Role of histone H3 lysine 9 trimethylation during bovine pre-implantation embryonic development

Histones play an important role in DNA’s compaction and organisation into the cellular nucleus. Depending on which histone modification occurs, chromatin can take a conformation of heterochromatin or euchromatin, which are associated with gene repression or expression, respectively. Histone H3 lysine 9 (H3K9) trimethylation (H3K9me3) is associated with gene silencing. At least 3 methyltransferases are able to change the methylation status of H3K9: SUV39H1, SUV39H2, and SETDB1. In several mammalian species, modulation of H3K9 methylation status has been demonstrated to be necessary to achieve a successful pre-implantation embryonic development after IVF or somatic cell NT. The aim of this work was to study the role of H3K9me3 in IVF pre-implantation bovine embryos. For this purpose, immunostaining of H3K9me3 at different pre-implantation stages of development was performed. Further, the relative abundances of the methyltransferases SUV39H1 and SUV39H2 were measured by real-time PCR using luciferase transcript as an exogenous gene for normalization. Finally, to evaluate H3K9me3 involvement during pre-implantation embryonic development, we generated SUV39H1 or SUV39H2 knockout embryos by the CRISPR/Cas9 system. We designed guide RNA targeting SUV39H1 or SUV39H2 and co-injected the presumptive zygote’s cytoplasm 18h post-fertilization with Cas9 protein. At Day 8 post-fertilization, the number of blastocysts was assessed and embryos were immunostained to evaluate H3K9me3. Results were analysed using Student’s t-test or ANOVA with the post-hoc Tukey test depending on data set (P ≤ 0.05) and reported as means±standard errors of the mean. Oocytes at germinal vesicle stage and metaphase II as well as embryos at different stages of pre-implantation development (2, 4, and 8 cells, morula, and blastocyst; n=6) were immunoreactive for H3K9me3. Expression of SUV39H1 and SUV39H2 mRNA decreased significantly as embryonic development progressed, reaching undetectable levels at stages where genome activation had already occurred (morula and blastocyst; P<0.0001, n=3). When zygotes were co-injected with the guide RNA targeting SUV39H1/Cas9, embryonic production showed a significant increase compared with the control [42.26%±5.03 (28/65) v. 23.86%±3.99 (21/88), respectively; P=0.034, n=4], and H3K9me3 immunostaining was reduced in treated embryos. Editing efficiency was estimated at 66%. In contrast, no statistical differences were found in embryonic production or H3K9me3 immunostaining in embryos co-injected with the guide RNA targeting SUV39H2/Cas9 (P=0.57, n=3). In conclusion, we were able to characterise H3K9me3 and determine transcript levels of methyltransferases SUV39H1 and SUV39H2 in oocytes and different stages of pre-implantation embryonic development. We also demonstrated that SUV39H1 deletion led to an increased embryonic production, suggesting that H3K9me3 removal would allow a greater relaxation of the heterochromatin and consequently a successful activation of embryonic genes. This highlights the essential role of H3K9me3 during bovine pre-implantation embryonic development.

Expression of endogenous retroviruses in pre‐implantation stages of bovine embryo

Endogenous retroviruses (ERVs) are involved in cellular proliferation, pluripotency, tissue-specific remodelling and regulation of developmental processes. These elements are transcriptionally active in mouse and human pre-implantation embryos. Empirical evidence indicates that regulatory networks involved with ERV transcripts are responsible for pluripotency and totipotency at certain stages of mouse and human pre-implantation development. Yet, the expression in pre-implantation bovine embryo remains unidentified. To determine whether two members of bovine endogenous retroviruses, BERV-K1 and BERV-K2, are expressed in the pre-implantation bovine embryo, each embryonic stage developed in vitro and was subjected to RNA release, reverse transcription and quantitative PCR. We found that BERV-K1 and BERV-K2 are expressed throughout different stages of pre-implantation development. The higher level of expression was detected in embryonic blastomeres with totipotent/pluripotent status (two-cell to 16-cell stages), while the more differentiated blastocyst stage showed significantly lower levels of ERVs expression. These findings suggest a possible role for endogenous retroviruses in the establishment of totipotent and pluripotent states in pre-implantation bovine embryo, similar to functions which have been suggested for these elements in human and mouse embryos.

The effects of mitochondrial DNA supplementation at the time of fertilization on the gene expression profiles of porcine preimplantation embryos.

Mitochondrial DNA (mtDNA) deficient metaphase II porcine oocytes are less likely to fertilize and more likely to arrest during preimplantation development. However, they can be supplemented with autologous populations of mitochondria at the time of fertilization, which significantly increases mtDNA copy number by the 2-cell stage due to the modulation of DNA methylation at a CpG island of the gene encoding the mtDNA-specific polymerase, POLG, and promotes preimplantation development. Although mitochondrial supplementation does not increase development rates or mtDNA copy number in oocytes with normal levels of mtDNA copy number, we tested whether this approach would also impact on chromosomal gene expression patterns in these oocytes at each stage of preimplantation development. Here, we have compared the gene expression profiles of embryos produced by mitochondrial supplementation at the time of fertilization with embryos produced by in vitro fertilization (IVF) using a panel of genes associated with different stages of preimplantation development. When compared to IVF-derived embryos, 27 (34%) genes were differentially expressed in supplemented embryos but this was restricted to one or two developmental stages. However, 53 (66%) genes were comparably expressed across all six stages and by the blastocyst stage 4 (5%) genes were differentially expressed. We conclude that additional copies of mtDNA can induce changes in gene expression at various stages of preimplantation development with the first changes occurring prior to maternal-to-zygotic transition (MZT). However, these changes appear to be transitory suggesting that some genomic resetting is taking place.

The Impact of Biopsy on Human Embryo Developmental Potential during Preimplantation Genetic Diagnosis.

Preimplantation Genetic Diagnosis and Screening (PGD/PGS) for monogenic diseases and/or numerical/structural chromosomal abnormalities is a tool for embryo testing aimed at identifying nonaffected and/or euploid embryos in a cohort produced during an IVF cycle. A critical aspect of this technology is the potential detrimental effect that the biopsy itself can have upon the embryo. Different embryo biopsy strategies have been proposed. Cleavage stage blastomere biopsy still represents the most commonly used method in Europe nowadays, although this approach has been shown to have a negative impact on embryo viability and implantation potential. Polar body biopsy has been proposed as an alternative to embryo biopsy especially for aneuploidy testing. However, to date no sufficiently powered study has clarified the impact of this procedure on embryo reproductive competence. Blastocyst stage biopsy represents nowadays the safest approach not to impact embryo implantation potential. For this reason, as well as for the evidences of a higher consistency of the molecular analysis when performed on trophectoderm cells, blastocyst biopsy implementation is gradually increasing worldwide. The aim of this review is to present the evidences published to date on the impact of the biopsy at different stages of preimplantation development upon human embryos reproductive potential.

CDNA microarray analysis of gene expression in parthenotes and in vitro produced buffalo embryos

The retarded development of parthenote embryo could be due to aberrant epigenetic imprinting, which may disrupt many aspects and lead to conceptus demise. The present work was conducted to: 1) compare the development of in vitro produced (IVP) and parthenogenetically developed (P) buffalo embryos from the 2-cell to blastocyst stage; 2) investigate the global gene expression profile and search for new candidate transcripts differing between IVP and P buffalo blastocyst using cDNA microarray analysis (validated by Real Time PCR); 3) follow the particular expression patterns of PLAC8 and OCT4 genes at five different stages of preimplantation development by Real Time PCR; and 4) study the expression of CDX2 at the blastcocyst stage. Cleavage rate was higher (P < 0.05) in P than IVP buffalo embryos, while, progression to blastocyst and number of cells per blastocyst were lower (P < 0.05) in P than IVP blastocysts. Microarray analysis indicate that 56 differentially expressed genes between the two groups, of which 51 genes (91.07%) were up-regulated, and five genes were downregulated in IVP blastocyst, using 1.4 fold-changes as a cutoff. Differentially expressed genes are related to translation, nucleic acid synthesis, cell adhesion and placentation. Validation of candidate genes revealed that the transcript abundance of PTGS2, RPS27A, TM2D3, PPA1, AlOX15, RPLO and PLAC8 were downregulated (7/8) in parthenote blastocyst compared to the IVP blastocyst. PLAC8 gene expression was higher (P < 0.05) at 2-cell, morula and blastocyst stages in IVP embryos compared with parthenote embryos. The OCT4 gene expression was higher (P < 0.05) in 2-cell, 4-cell, 8-cell and blastocysts produced in vitro. In conclusion, the retarded development of parthenogenetic buffalo embryos could be due to downregulation of genes related to translation, nucleic acid synthesis, cell adhesion, and placental development. The low expression of PLAC8 and OCT4 during the different stages of development may be responsible, in part, to the failure of development of parthenote buffalo embryos.

Lipocalin-1: a potential marker for noninvasive aneuploidy screening

This study has identified the first protein, lipocalin-1, in the secretome of human blastocysts that is associated with chromosome aneuploidy. The development of a noninvasive technique to determine an embryo's developmental competence, including chromosomal constitution, by analysis of spent IVF culture medium will be a powerful tool for embryo selection in assisted reproductive technologies.

Housekeeping Gene Transcript Abundance in Bovine Fertilized and Cloned Embryos

The objective of this study was to compare housekeeping gene expression levels, relative to total mRNA, across different stages of bovine preimplantation development in embryos generated by IVF and somatic cell nuclear transfer (SCNT). We first analyzed the levels of total RNA recovered from different stages of preimplantation development. A similar RNA level was observed from oocytes to 16-cell stage embryos with a significant increase at morula and blastocyst stages. Then we used an absolute mRNA determination method that accounts for the RNA level in the embryo by quantifying copies of transcripts normalized to loaded cDNA amount. The number of housekeeping genes mRNA copies per nanogram of cDNA was compared among samples obtained from different stages of preimplantation IVF-derived embryos. None of the genes analyzed (GAPDH, PPIA, ACTB, RPL15, GUSB, and Histone H2A.2) maintained constant levels throughout preimplantation development, indicating that they are not suitable for normalizing gene expression across developmental stages. We then compared expression of housekeeping genes between IVF and SCNT embryos at different embryonic stages. We found different levels of transcript abundance between IVF and SCNT embryos for GAPDH, RPL15, GUSB, and ACTB. On the other hand, Histone H2A.2 and PPIA were similar between IVF and SCNT embryos at each stage analyzed, although they varied across stages as previously mentioned.

Micromanipulation Techniques in IVF

After the world witnessed groundbreaking early advances in assisted reproduction that utilized conventional in vitro fertilization in the human, more complex invasive techniques of oocyte and embryo manipulation were introduced to treat human subfertility. Amongst these were numerous micromanipulation techniques such as partial zona dissection (PZD; 1988), subzonal insemination (SUZI, 1988) and subsequently ICSI, which became the most powerful tool of assisted fertilization (1992). ICSI requires micromanipulation of both the male and the female gamete. Another important approach is laser-assisted opening of the zona pellucida at different stages of preimplantation development either; to assist hatching (1990) or to remove cells for preimplantation genetic diagnosis (1989), or fragments to restore the integrity of an embryo (1999). Sometimes even blastocysts considered for vitrification need further manipulation in order to artificially shrink the blastocoel which facilitates survival of expanded blastocyst (2002). Other methods for reproductive purposes, however, failed to gain acceptance in population, such as cytoplasm transfer (1997), nuclear transfer (1998) and cloning (1996), to name but a few.

Oocyte maturation failure: a syndrome of bad eggs

To show that disruption of meiotic competence results in cell cycle arrest, and the production of immature oocytes that are not capable of fertilization. Through an extensive review of animal studies and clinical case reports, we define the syndrome of oocyte maturation failure as a distinct oocyte disorder, present a classification system based on clinical parameters, and discuss the potential molecular origins for the disease.

Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al.1 and Wilmut 2 separately described that mouse embryos could be frozen and stored at -196 °C and, a few years later, Parkening et al. 3 reported the birth of live offspring resulting from in vitro fertilization (IVF) of cryopreserved oocytes. Since then, the use of cryopreservation techniques has rapidly spread to become an essential component in the practice of human and animal assisted reproduction and in the conservation of animal genetic resources. Currently, there are two main methods used to cryopreserve oocytes and embryos: slow freezing and vitrification. A wide variety of approaches have been used to try to improve both techniques and millions of animals and thousands of children have been born from cryopreserved embryos. However, important shortcomings associated to cryopreservation still have to be overcome, since ice-crystal formation, solution effects and osmotic shock seem to cause several cryoinjuries in post-thawed oocytes and embryos. Slow freezing with programmable freezers has the advantage of using low concentrations of cryoprotectants, which are usually associated with chemical toxicity and osmotic shock, but their ability to avoid ice-crystal formation at low concentrations is limited. Slow freezing also induces supercooling effects that must be avoided using manual or automatic seeding 4. In the vitrification process, high concentrations of cryoprotectants inhibit the formation of ice-crystals and lead to the formation of a glasslike vitrified state in which water is solidified, but not expanded. However, due to the toxicity of cyroprotectants at the concentrations used, oocytes/embryos can only be exposed to the cryoprotectant solution for a very short period of time and in a minimum volume solution, before submerging the samples directly in liquid nitrogen 5. In the last decade, vitrification has become more popular because it is a very quick method in which no expensive equipment (programmable freezer) is required. However, slow freezing continues to be the most widely used method for oocyte/embryo cryopreservation. In this video-article we show, step-by-step, how to collect and slowly freeze hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

Derivation of germ-line-competent embryonic stem cell lines from preblastocyst mouse embryos.

The first differentiation event of the mammalian embryo is thought to occur during blastulation and results in two populations of cells, the inner cell mass (ICM) and the trophectoderm. Most embryonic stem (ES) cell lines have been derived from the ICM or a further subset of ICM cells known as the epiblast. There appears to be a limited period of embryonic development during which pluripotent ES cells can be adapted from the cells of the blastocyst to culture. A method is presented here that allows ES cell lines to be isolated from preblastocyst mouse embryos. These lines were derived from 129S2/SvHsd mouse morulae and earlier cleavage stages with high efficiency. The lines expressed genes and antigens characteristic of pluripotent ES cells. XY cell lines remained karyotypically stable through extensive passaging and produced germ-line-competent chimeras upon blastocyst injection. These results suggest that true ES cells can be derived from embryos explanted at any stage of preimplantation development in the mouse. This finding raises the interesting question of whether ES cell lines derived from embryos at different stages of preimplantation development possess the same potential.

Matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase expression in human preimplantation embryos

Objective To examine human embryos at various stages of preimplantation development for simultaneous expression of matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases (TIMPs). Design mRNAs of specific MMPs and TIMPs were examined in single human embryos, at different stages of preimplantation development, by reverse transcription-polymerase chain reaction (RT-PCR). Single embryo immunohistochemistry was applied to examine the protein expression. Setting University-affiliated IVF-ET program. Patient(s) Couples, attending the university-affiliated IVF-ET program, electing to donate poor prognosis embryos with anomalous morphology. Intervention(s) None. Main outcome measure(s) The expression of MMP-1, MMP-2, MMP-9, TIMP-1, TIMP-2, and TIMP-3 in preimplantation embryos. Result(s) The MMP-2 mRNA was expressed consistently during development from one-cell to blastocyst stage. The TIMP-1 and TIMP-3 mRNAs were detected in embryos at all stages; however, in the later preimplantation developmental stages, an increasing proportion of embryos expressing TIMP-1 and TIMP-3 mRNA were noted. The MMP-1, MMP-9, and TIMP-2 mRNAs were detected in only a minority of human embryos studied. Immunohistochemistry showed MMP-1 and TIMP-1 protein expression in preimplantation embryos. Conclusion(s) The existence of MMP and TIMP mRNA expression in human preimplantation embryos argues for a role for these metalloproteinases and their inhibitors during the process of implantation in humans.

Pronuclear morphology evaluation with subsequent evaluation of embryo morphology significantly increases implantation rates

Objective To elucidate the relative predictive value of implantation markers at different stages of preimplantation development. Design Correlation of pronuclear morphology with embryo morphology and implantation rates in retrospective and prospective analysis of in vitro fertilization/intracytoplasmic sperm injection (IVF-ICSI) treatment cycles. Setting Private infertility center. Patient(s) A total of 441 couples undergoing infertility treatment. Intervention(s) None. Main outcome measure(s) Size of pronuclei and distance between them, the number and polarization of nucleolus precursor bodies (NPB) at the one-cell stage, embryo cleavage and fragmentation rates on days 2 and 3, and pregnancy and implantation rates. Result(s) Polarization of the NPB in both pronuclei had a statistically significant correlation with normal membrane breakage during ICSI (40%, compared with 33% easy, and 31% difficult membrane breakage) and also with faster cleavage and lower fragmentation rates of embryos. Sixty-one percent of implanting embryos had polarization of the NPB in both pronuclei compared with 37% for all embryos. Larger distance between pronuclei and their unequal size had a statistically significant correlation with slower cleavage and inferior embryo quality. Embryo selection based on only pronuclear morphology or on only day-3 embryo morphology yielded implantation rates of 15.1% and 12.1%, respectively. Embryo selection based on sequential evaluation of both pronuclear morphology and embryo morphology on day 3 resulted in a 21.1% implantation rate. Conclusion(s) Polarization of NPB in both pronuclei is as reliable marker of implantation as embryo morphology on day 3. However, pronuclear morphology assessment improves embryo selection only when it is combined with embryo morphology evaluation on day 3.

Selection based on morphological assessment of oocytes and embryos at different stages of preimplantation development: a review.

In contrast to IVF, in ICSI the surrounding cumulus and corona cells must be removed completely, as only denuded oocytes can be successfully manipulated by the holding pipette. This ancillary effect of ICSI allows us to focus on the morphology of preimplantation development from the earliest stages. Early prognosis regarding the developmental fate of oocytes would help to limit a negative impact of culture conditions. However, little evidence is available that non-invasive selection at the oocyte stage (first polar body, granular cytoplasm) may be of prognostic value. Recently, certain patterns of pronuclei (number and the distribution of nucleoli) at the zygote stage were found to correlate with treatment outcome in IVF and ICSI cycles, offering an additional prognostic tool prior to cleavage. As there is evidence that embryo selection on day 2 or 3 based on morphological criteria (fragmentation, number of blastomeres, multinucleation, uneven cleavage) may be imprecise, patients might benefit from extended embryo culture to day 5. However, not all major chromosomal aberrations are incompatible with blastocyst formation, and prolonged culture in vitro does not exclusively select embryos with a normal chromosomal complement. Consequently, special care should be taken to minimize the presence of aneuploid concepti in culture. In addition, multiple selection at different stages of development will be required to filter out the correct 'candidate' embryo which will result in a healthy newborn.

Analysis of human preimplantation embryos and aging oocytes by cDNA microarrays

Objective: To develop methods to analyze single oocytes and preimplantation embryos by cDNA microarrays 1) to define differences between oocytes from younger and older reproductive-aged women, and 2) to examine the cascade of gene expression that follows fertilization. Design: Descriptional study of human oocyte and embryo gene expression. Materials/Methods: These studies were approved by the UCSF Committee on Human Research. Consenting patients from the UCSF in vitro fertilization (IVF) program were candidates for these studies. Donated immature (germinal vesicle) oocytes from patients undergoing ICSI were stripped of granulosa cells and frozen individually in tubes. Embryos were obtained from patients who had completed their families and wished their remaining embryos be donated to research rather than destroyed. RNA was prepared using PicoPure RNA Isolation Kit (Arcturus Engineering), and amplified using a high sensitivity proprietary kit (collaborative effort, Arcturus Engineering). Cy3 (control) and Cy5 fluorescently-labeled probes were reverse-transcribed from amplified RNA. Two microarray chips (∼22,000 genes and ESTs each) were hybridized, washed, and scanned using the GenePix 4000B scanner. To test the amplification protocol, RNA from two oocytes was mixed then divided into two tubes for independent amplification. Analysis was performed using the Pearson Correlation. Oocytes from women 30 years and less were compared to oocytes from women 40 years and older, and to embryos at day 1 (pronuclear) and day 3 of development. Analysis was performed using GenePix 3.0, Significance Analysis of Microarrays (Stanford), and Cluster/TreeView (Lawrence Berkeley National Lab) software. Results: RNA amplification using these techniques was 1.8 million-fold, sufficient for multiple microarray experiments. The amplification control yielded a correlation coefficient of 98.3%, demonstrating reproducibility of the amplification methods. SAM analysis identified the most differentially expressed genes; more importantly, however, it revealed that biological and experimental variability can be overcome by relatively few experiments. Few genes are differentially expressed between young and old oocytes, suggesting that any differences are likely functional or at the protein level. Striking differences are evident by day 1 of embryo development, and this pattern contrasts greatly with those at day 3. Cluster analysis clearly demonstrates unique expression profiles among the different stages of development tested and has identified potential candidate genes for further study. Conclusions: cDNA microarray analysis of extremely small samples is now technologically feasible. These studies have shown few differences in the RNA between young and old eggs, but robust changes at the pronuclear stage of embryo development following fertilization. Future studies will more fully define the differences between the oocytes of younger and older women, and the different stages of preimplantation development. Supported by: Reproductive Scientist Development Program and Arcturus Engineering (amplification kits).

Estrogen receptor is expressed in pig embryos during preimplantation development.

Although estrogen is recognized as essential for embryonic development and maintenance of pregnancy, it remains unclear whether it has a direct role in the embryos themselves. The aim of this study was to investigate whether estrogen can have any effect in pig embryos during preimplantation development. Since the function of estrogen is mediated through its specific receptor, estrogen receptor (ER), the presence of ER mRNA and protein in pig embryos collected in vivo at different stages of preimplantation development was determined and compared. Using reverse transcription polymerase chain reaction, ER RNA was detected at the one-cell, two-cell, and four-cell stages. The level became undetectable at the five- to eight-cell stages and the morula stages and then reappeared again at the blastocyst stage. To determine whether the ER message observed in the embryos was translated into ER protein, immunocytochemical analysis was performed and the presence of ER protein was detected in oocytes at one-cell and four-cell stages. However, the amount of ER protein in porcine embryos at the blastocyst stage was still below the detection limit. The presence of ER mRNA at the blastocyst stages suggests that estrogen may start to act directly on pig embryos afterwards, and our results provide a basis for determining the direct role of estrogen in preimplantation pig embryos.

Estrogen receptor is expressed in pig embryos during preimplantation development

Although estrogen is recognized as essential for embryonic development and maintenance of pregnancy, it remains unclear whether it has a direct role in the embryos themselves. The aim of this study was to investigate whether estrogen can have any effect in pig embryos during preimplantation development. Since the function of estrogen is mediated through its specific receptor, estrogen receptor (ER), the presence of ER mRNA and protein in pig embryos collected in vivo at different stages of preimplantation development was determined and compared. Using reverse transcription polymerase chain reaction, ER RNA was detected at the one-cell, two-cell, and four-cell stages. The level became undetectable at the five- to eight-cell stages and the morula stages and then reappeared again at the blastocyst stage. To determine whether the ER message observed in the embryos was translated into ER protein, immunocytochemical analysis was performed and the presence of ER protein was detected in oocytes at one-cell and four-cell stages. However, the amount of ER protein in porcine embryos at the blastocyst stage was still below the detection limit. The presence of ER mRNA at the blastocyst stages suggests that estrogen may start to act directly on pig embryos afterwards, and our results provide a basis for determining the direct role of estrogen in preimplantation pig embryos. Mol. Reprod. Dev. 55:83–88, 2000. © 2000 Wiley-Liss, Inc.

Paternal transcripts for glucose-6-phosphate dehydrogenase and adenosine deaminase are first detectable in the human preimplantation embryo at the three- to four-cell stage.

The transition between dependence on maternal transcripts and proteins inherited in the oocyte and embryonic gene expression in the human preimplantation embryo occurs at the four- to eight-cell stage. Recently, studies using reverse transcriptase polymerase chain reaction (RT-PCR) have detected paternal transcripts for the Y-linked genes, ZFY and SRY, and the myotonic dystrophy associated protein kinase gene, DK, as early as the late pronucleate one-cell stage. However, expression at the protein level has not been demonstrated and its function at these early stages is unknown. Using coding sequence polymorphisms to distinguish maternal and paternal transcripts, we have examined the transcription of two ubiquitously expressed genes: X-linked glucose-6-phosphate dehydrogenase (G6PD) and adenosine deaminase (ADA). Both G6PD and ADA are housekeeping genes with TATA-less promoters which, because of their roles in metabolism and ubiquitous expression, may provide a more reliable indication of the timing of activation of the embryonic genome. They also each have biallelic polymorphisms with a high heterozygosity ratio which can be detected by restriction digestion. Couples undergoing in vitro fertilization (IVF) were screened for these polymorphisms. Individual spare oocytes and embryos at different stages of preimplantation development were analyzed by RT-PCR and appropriate restriction digestion in those cases in which the male partner carried a different allele to the female partner. In addition, since only female embryos inherit the paternal allele of X-linked G6PD, cDNA was also analyzed for ZFX/ZFY transcripts to identify the sex of each embryo. One hundred and twenty three individual oocytes and embryos were analyzed by RT-PCR and restriction digestion to detect the paternal transcripts from the polymorphic alleles. Maternal transcripts for G6PD, ADA, and ZFX were detected in all oocytes and embryos and at all stages. Following restriction digestion, paternal G6PD and ZFY transcripts were first detected at the four-cell stage and paternal ADA transcripts in an embryo at the three-cell stage coinciding with the onset of dependency on transcription from the embryonic genome. This approach should be widely applicable to other genes since similar polymorphisms exist in the coding regions of many genes.