Mechanistic insights into zinc oxide nanoparticles induced embryotoxicity via H3K9me3 modulation.

The widespread application of nanoparticles (NPs) in various fields has raised health concerns, especially in reproductive health. Our research has shown zinc oxide nanoparticles (ZnONPs) exhibit the most significant toxicity to pre-implantation embryos in mice compared to other common NPs. In patients undergoing assisted reproduction technology (ART), a significant negative correlation was observed between Zn concentration and clinical outcomes. Therefore, this study explores the impact of ZnONPs exposure on pre-implantation embryonic development and its underlying mechanisms. We revealed that both in vivo and in vitro exposure to ZnONPs impairs pre-implantation embryonic development. Moreover, ZnONPs were found to reduce the pluripotency of mouse embryonic stem cells (mESCs), as evidenced by teratoma and diploid chimera assays. Employing multi-omics approaches, including RNA-Seq, CUT&Tag, and ATAC-seq, the embryotoxicity mechanisms of ZnONPs were elucidated. The findings indicate that ZnONPs elevate H3K9me3 levels, leading to increased heterochromatin and consequent inhibition of gene expression related to development and pluripotency. Notably, Chaetocin, a H3K9me3 inhibitor, sucessfully reversed the embryotoxicity effects induced by ZnONPs. Additionally, the direct interaction between ZnONPs and H3K9me3 was verified through pull-down and immunoprecipitation assays. Collectively, these findings offer new insights into the epigenetic mechanisms of ZnONPs toxicity, enhancing our understanding of their impact on human reproductive health.

Expression of miRNA from spent pre-implantation embryos culture media.

This study examines the expression of three microRNAs (hsa-miR-661, hsa-miR-21-5p, hsa-miR-372-5p) in spent pre-implantation embryos culture media to identify possible new non-invasive biomarkers of embryo competence, predictive of development to the blastocyst stage. A preliminary analysis on 16 patients undergoing IVF cycles was performed by collecting and stored spent culture media on the fifth/sixth day of embryo culture. Expression of miRNAs was evaluated according to the embryos' fate: 1) NE/DG: non-evolved or degenerate embryos; 2) BLOK: embryos developed to the blastocyst stage. Preliminary results revealed a higher miRNAs expression in NE/DG spent media. To elucidate the roles of these miRNAs, we employed a robust bioinformatics pipeline involving: 1) in-silico miRNA Target Prediction using RNAHybrid, which identified the most-likely gene targets; 2) Construction of a Protein-Protein Interaction network via GeneMania, linking genes with significant biological correlations; 3) application of modularity-based clustering with the gLay app in Cytoscape, resulting in three size-adapted subnets for focused analysis; 4) Enrichment Analysis to discern the biological pathways influenced by the miRNAs. Our bioinformatics analysis revealed that hsa-miR-661 was closely associated with pathways regulating cell shape and morphogenesis of the epithelial sheet. These data suggest the potential use of certain miRNAs to identify embryos with a higher likelihood of developing to the blastocyst stage. Further analysis will be necessary to explore the reproducibility of these findings and to understand if miRNAs here investigated can be used as biomarkers for embryo selection before implantation into the uterus or if they may be reliable predictors of IVF outcome.

TOP2A deficiency leads to human recurrent spontaneous abortion and growth retardation of mouse pre-implantation embryos.

BACKGROUND: Recurrent spontaneous abortion (RSA), is a dangerous pregnancy-related condition and is a subject of debate in the gynaecology and obstetrics communities. The objective of this study was to determine the function of DNA Topoisomerase II Alpha (TOP2A) in RSA and elucidate the underlying molecular mechanisms. METHODS: In vitro models of TOP2A-knockdown and -overexpression were generated by transfecting specific sh-RNA lentivirus and overexpression plasmid, respectively. An in vitro TOP2A inhibition model was established by culturing mouse embryos at the two-cell stage in a medium containing PluriSIn2, a TOP2A inhibitor. Immunohistochemical staining was used to analyse expression of TOP2A in villi tissues of patients with RSA. Western blotting and qRT-PCR were used to analyse the expression of TOP2A and proteins involved in trophoblast functions, the FOXO signalling pathway, and the development of pre-implantation embryos. 5-Ethynyl-2'-deoxyuridine staining, TUNEL assay and flow cytometry were used to further evaluate the effect of TOP2A on cell proliferation and apoptosis. Transwell and wound healing assays were used to evaluate migration and invasion. Moreover, the effect of TOP2A inhibitor on embryos was determined by immunofluorescence and mitochondrial-related dyes. RESULTS: Evaluation of clinical samples revealed that the villi tissues of patients that have experienced RSA had lower TOP2A expression compared with that from women who have experienced normal pregnancy (P < 0.01). In vitro, TOP2A knockdown decreased the proliferation, migration, and invasion of trophoblast cell lines, and increased apoptosis and activation of the FOXO signalling pathway (P < 0.05). Conversely, TOP2A overexpression reversed these effects. Moreover, in vivo experiments confirmed that inhibition of TOP2A impairs trophectoderm differentiation, embryonic mitochondrial function as well as the developmental rate; however, no differences were noted in the expression of zygotic genome activation-related genes. CONCLUSIONS: Collectively, our data suggest that lower TOP2A expression is related to RSA as it inhibits trophoblast cell proliferation, migration, and invasion by activation of the FOXO signalling pathway. Additionally, TOP2A inhibition resulted in impaired development of pre-implantation embryos in mice, which could be attributed to excessive oxidative stress.

2,4,6-triiodophenol exhibits embryotoxicity to pre-implantation mouse embryos in an in vitro exposure model.

2,4,6-triiodophenol (TIP), a novel type of halophenolic disinfection byproducts, has been widely detected in water bodies, even in drinking water. Recently, TIP has drawn increasing concerns on account of considerable developmental toxicity towards lower organisms and cytotoxicity for mammalian cells. However, it remains unknown about its toxicity on mammalian pre-implantation embryos. Here, by exposing mouse zygotes derived in vitro fertilization to TIP, which ranged from 5 to 50 µM, we found that TIP impaired the quality of pre-implantation mouse embryos in a dose-dependent manner, inducing decline of both total and trophectoderm cell numbers, enhancing caspase 3/7 activity and reactive oxygen species generation, though it did not decrease blastocyst formation efficiency. For the sake that only high qualified embryos are able to implant in endometrium and generate health body finally, we applied a previously modified in vitro culture system to assess TIP-exposed blastocysts' further developmental potency beyond pre-implantation stage. Surprisingly, although the exposed dose was only 5 µM and TIP was removed as soon as the zygotes reached blastocyst stage, these blastocysts still nearly lost their implantation and egg cylinder formation ability, exhibiting abnormal embryonic lineage differentiation pattern as well. Therefore, our study not only entirely shows TIP embryonic toxicity on mouse pre-implantation embryos, but also proposes a model to evaluate embryotoxicity from the zygote to egg cylinder stage.

Distinct role of histone chaperone Asf1a and Asf1b during fertilization and pre-implantation embryonic development in mice.

BACKGROUND: Asf1 is a well-conserved histone chaperone that regulates multiple cellular processes in different species. Two paralogous genes, Asf1a and Asf1b exist in mammals, but their role during fertilization and early embryogenesis remains to be investigated further. METHODS: We analyzed the dynamics of histone chaperone Asf1a and Asf1b in oocytes and pre-implantation embryos in mice by immunofluorescence and real-time quantitative PCR, and further investigated the role of Asf1a and Asf1b during fertilization and pre-implantation development by specific Morpholino oligos-mediated knock down approach. RESULTS: Immunofluorescence with specific antibodies revealed that both Asf1a and Asf1b were deposited in the nuclei of fully grown oocytes, accumulated abundantly in zygote and 2-cell embryonic nuclei, but turned low at 4-cell stage embryos. In contrast to the weak but definite nuclear deposition of Asf1a, Asf1b disappeared from embryonic nuclei at morula and blastocyst stages. The knockdown of Asf1a and Asf1b by specific Morpholino oligos revealed that Asf1a but not Asf1b was required for the histone H3.3 assembly in paternal pronucleus. However, knockdown of either Asf1a or Asf1b expression decreased developmental potential of pre-implantation embryos. Furthermore, while Asf1a KD severely reduced H3K56 acetylation level and the expression of Oct4 in blastocyst stage embryos, Asf1b KD almost eliminated nuclear accumulation of proliferating cell marker-PCNA in morula stage embryos. These results suggested that histone chaperone Asf1a and Asf1b play distinct roles during fertilization and pre-implantation development in mice. CONCLUSIONS: Our data suggested that both Asf1a and Asf1b are required for pre-implantation embryonic development. Asf1a regulates H3K56ac levels and Oct4 expression, while Asf1b safeguards pre-implantation embryo development by regulating cell proliferation. We also showed that Asf1a, but not Asf1b, was necessary for the assembly of histone H3.3 in paternal pronuclei after fertilization.

The challenge of getting a high quality of RNA from oocyte for gene expression study.

The extraction of intact RNA from oocyte is quite challenging and time-consuming. A standard protocol using commercial RNA extraction kit, yields a low quantity of RNA in oocytes. In the past, several attempts in getting RNA for gene expression study ended up with a few different modified methods. Extraction of high-quality RNA from oocyte is important before further downstream analyses such as reverse transcription-polymerase chain reaction, quantitative polymerase chain reaction, or northern blot analysis. In this review, the efficiency of RNA extraction methods from all species oocytes was compared between published articles and our research to gather all possible methods of RNA extraction. Two different methods of RNA extraction that were proposed from various experiments were reviewed to determine the best method of RNA extraction from the oocyte. Modified TRIzol method can be concluded as an efficient RNA extraction method especially for good RNA from oocytes. Meanwhile, comparing RNA extraction kits to extract the RNA from oocytes or pre-implantation embryos, the micro RNA extraction kit type is the best. Therefore, an appropriate RNA extraction method is important to obtain high quality of total RNA for gene expression profiling analysis.

Heterochromatin Morphodynamics in Late Oogenesis and Early Embryogenesis of Mammals.

During the period of oocyte growth, chromatin undergoes global rearrangements at both morphological and molecular levels. An intriguing feature of oogenesis in some mammalian species is the formation of a heterochromatin ring-shaped structure, called the karyosphere or surrounded "nucleolus", which is associated with the periphery of the nucleolus-like bodies (NLBs). Morphologically similar heterochromatin structures also form around the nucleolus-precursor bodies (NPBs) in zygotes and persist for several first cleavage divisions in blastomeres. Despite recent progress in our understanding the regulation of gene silencing/expression during early mammalian development, as well as the molecular mechanisms that underlie chromatin condensation and heterochromatin structure, the biological significance of the karyosphere and its counterparts in early embryos is still elusive. We pay attention to both the changes of heterochromatin morphology and to the molecular mechanisms that can affect the configuration and functional activity of chromatin. We briefly discuss how DNA methylation, post-translational histone modifications, alternative histone variants, and some chromatin-associated non-histone proteins may be involved in the formation of peculiar heterochromatin structures intimately associated with NLBs and NPBs, the unique nuclear bodies of oocytes and early embryos.

Primed Track: Reliable Volumetric Single-cell Tracking and Lineage Tracing of Living Specimen with Dual-labeling Approaches.

Mammalian embryonic development starts with a single fertilized zygote that develops into a blastocyst embryo consisting of three cell types that evolve into either embryonic or extra-embryonic tissues. Lineage tracing of these cells can provide important information about the molecular and cellular dynamics contributing to fate allocation during early development. While global labeling techniques allow for visualization of all cells at the same time, lineage tracing of cells over several divisions can become complicated due to embryo movement and rotation as well as increasing cell densities. Here, we use green-to-red photoconvertible proteins for both global and sparse labeling of cells of interest in the developing murine embryo. We use primed conversion to achieve precise photoconversion of single nuclei in 4-cell stage embryos followed by volumetric live imaging to capture development up to the blastocyst stage. We developed an image analysis pipeline, called primed Track, that uses the dual labeling strategy for both straightforward segmentation and registration of all cells in the embryo as well as correction of rotational and spatial drift. Together, this strategy allows for reliable and fast tracking and lineage tracing of individual cells, even over increased imaging time intervals that result in a major reduction in data volume, all essential conditions for volumetric long-term imaging techniques.

Colony aging affects the reproductive performance of Swiss Webster females used as recipients for embryo transfer.

The objective was to evaluate the influence of colony aging in a Swiss Webster (SW) outbred stock used as recipients for embryo transfer. In the first study, a retrospective analysis was performed throughout several generations during a 38-month period in 2,398 embryos transferred to 108 SW recipients. A decrease in the percentage of live pups from transferred embryos was found at the end of the period. Impairment occurred due to the incidence of maternal cannibalism that increased from 0% to 67-100% (P<0.05), while pregnancy rate (pregnant/transferred recipients) and number of pups per delivered female were not affected throughout the period (P=NS). A following study was carried out to compare the reproductive performance of SW stock vs. B6D2F1 hybrid females in a 5-year interval. The study was conducted on a total of 893 embryos transferred to 40 females (20 SW and 20 B6D2F1) in Year #1, and 514 embryos transferred to 30 females (15 SW and 15 B6D2F1) in Year #5. No cases of maternal cannibalism were found on Year #1 in any of the strains (0/10 and 0/10). However, an incidence of 44,4% (4/9) was seen on Year #5 for SW, while for B6D2F1 the incidence was 0% (0/12) (P<0.05). Further examination of the uterus showed endometrial cysts and abnormal implantation sites in SW on Year #5 but not in B6D2F1 females. In conclusion, this study reports an impairment of the reproductive performance of an early aged SW outbred stock colony mainly due to the occurrence of maternal cannibalism. This finding has important implications for embryo transfer programs conducted in mouse facilities.

Functions of intrinsic disorder in proteins involved in DNA demethylation during pre-implantation embryonic development.

DNA demethylation is involved in many biological processes during pre-implantation embryonic development in mammals. To date, the complicated mechanism of DNA demethylation is still not fully understood. Ten-eleven translocation family (TET3, TET1 and TET2), thymine DNA glycosylase (TDG) and DNA methyltransferase 1 (DNMT1) are considered the major protein enzymes of DNA demethylation in pre-implantation embryos. TET3, TET1, TET2, TDG, and DNMT1 contain abundant levels of intrinsically disordered protein regions (IDPRs), which contribute to increasing the functional diversity of proteins. Thus we tried to explore the complicated DNA demethylation in pre-implantation embryos from the intrinsic disorder perspective. These five biological macromolecules all have DNA demethylation-related functional domains. They can work together to fulfill DNA demethylation in pre-implantation embryos through complex protein-protein interaction networks. Intrinsic disorder analysis results showed these proteins were partial intrinsically disordered proteins. Many identifiable disorder-based DNA-binding sites, protein-binding sites and post-translational modification sites located in the intrinsically disordered regions, and DNA demethylation deficiency point mutations in the IDPRs could significantly change the local disorder propensity of these proteins. To the best of our knowledge, this work provides a new viewpoint for studying the mechanism of DNA methylation reprogramming during mammalian pre-implantation embryonic development.

Characterization and comparative analyses of transcriptomes of cloned and in vivo fertilized porcine pre-implantation embryos.

Somatic cell nuclear transfer (SCNT) is the only method known to rapidly reprogram differentiated cells into totipotent embryos. Most cloned embryos become arrested before implantation and the details of the underlying molecular mechanism remain largely unknown. Dynamic regulation of the transcriptome is a key molecular mechanism driving early embryonic development. Here, we report comprehensive transcriptomic analysis of cloned embryos (from Laiwu and Duroc pigs) and in vivo fertilized embryos (from Duroc pigs) using RNA-sequencing. Comparisons between gene expression patterns were performed according to differentially expressed genes, specific-expressed genes, first-expressed genes, pluripotency genes and pathway enrichment analysis. In addition, we closely analyzed the improperly expressed histone lysine methyltransferases and histone lysine demethylases during cell reprogramming in cloned embryos. In summary, we identified altered gene expression profiles in porcine cloned pre-implantation embryos in comparison to normal in vivo embryos. Our findings provide a substantial framework for further discovery of the epigenetic reprogramming mechanisms in porcine SCNT embryos.

DNA methylation, environmental exposures and early embryo development.

The first crucial step in the developmental program occurs during pre-implantation, the time after the oocyte has been fertilized and before the embryo implants in the uterus. This period represents a vulnerable window as the epigenome undergoes dynamic changes in DNA methylation profiles. Alterations in the early embryonic reprogramming wave can impair DNA methylation patterns and induce permanent changes to the developmental program, leading to the onset of adverse health outcomes in offspring. Although there is an increasing body of evidence indicating that harmful exposures during pre-implantation embryo development can trigger lasting epigenetic alterations in offspring, the mechanisms are still not fully understood. Since physiological or pathological changes in DNA methylation can occur as a response to environmental cues, proper environmental milieu plays a critical role in the success of embryonic development. In this review, we depict the mechanisms behind the embryonic epigenetic reprogramming of DNA methylation and highlight how maternal environmental stressors (e.g., alcohol, heat stress, nutrient availability) during pre-implantation and assisted reproductive technology procedures affect development and DNA methylation marks.

Expression and distribution of the zinc finger protein, SNAI3, in mouse ovaries and pre-implantation embryos.

The Snail gene family includes Snai1, Snai2, and Snai3 that encode zinc finger-containing transcriptional repressors in mammals. The expression and localization of SNAI1 and SNAI2 have been studied extensively during folliculogenesis, ovulation, luteinization, and embryogenesis in mice. However, the role of SNAI3 is unknown. In this study, we investigated the expression of SNAI3 during these processes. Our immunohistochemistry data showed that SNAI3 first appeared in oocytes by postnatal day (PD) 9. Following this, SNAI3 was found to be expressed consistently in theca and interstitial cells, along with oocytes. In gonadotropin-treated immature mice, the expression of SNAI3 did not change significantly during follicular development. The expression of SNAI3 was reduced during ovulation, after which it increased gradually during luteinization. Similar results were obtained from western blot analyses. Furthermore, real-time polymerase chain reaction (RT-PCR) analyses revealed varying mRNA levels of different Snail factors at a given time in gonadotropin-induced ovaries. During early embryo cleavage, SNAI3 was localized to the nucleus, except the nucleolus at the germinal vesicle and one-cell stages. From two- to eight-cell stages, SNAI3 was localized only to the nucleolus. Thereafter, SNAI3 was detected only in the cytoplasm, except during the blastocyst stage when it was localized to the nucleus of the trophectoderm and the inner cell mass. RT-PCR results showed that the expression of Snail superfamily genes was decreased during the blastocyst stage. From the eight-cell to morula stage, when compaction occurs that is a prerequisite for blastocyst formation, Snai3 mRNA was expressed at very low levels and was opposite to the highest expression level of the compaction-related gene, E-cadherin, at the eight-cell stage. Taken together, our results suggest that SNAI3 likely plays some roles during folliculogenesis, luteinization, and early embryonic development.

Expression and distribution of forkhead activin signal transducer 2 (FAST2) during follicle development in mouse ovaries and pre-implantation embryos.

Xenopus forkhead activin signal transducer 1 (xFAST 1) was first characterized in Xenopus as the transcriptional partner for Smad proteins. FAST2, which is the xFAST 1 homologues in mouse, is expressed during early developmental stages of the organism. However, the function of FAST2 in mouse ovaries and pre-implantation embryos is unclear. Therefore, we investigated its expression during these processes. In postnatal mice, FAST2 was expressed in oocytes and thecal cells from postnatal day (PD) 1 to PD 21. In gonadotropin-induced immature mice, FAST2 was expressed in oocytes, thecal cells and newly formed corpora lutea (CLs), but was expressed at a lower level in degenerated CLs. Similar results were observed upon western blot analyses. In meloxicam-treated immature mice, ovulation was inhibited and FAST2 was expressed in thecal cells, luteinized granulosa cells and entrapped oocytes. Immunofluorescence results showed that FAST2 was expressed in the cytoplasm and nucleus but not the nucleolus from the zygote to 8-cell embryo stage, after which it was localized to the cytoplasm of the morulae and inner cell mass of the blastocysts. Taken together, these observations suggest that FAST2 is expressed in a cell-specific manner during ovarian follicle development, ovulation, luteinization and early embryonic development, and that FAST2 might play important roles in these physiological processes.

Tracing the expression of circular RNAs in human pre-implantation embryos.

BACKGROUND: PolyA- RNAs have not been widely analyzed in human pre-implantation embryos due to the scarcity of materials. In particular, circular RNA (circRNA), a novel type of polyA- RNA, has not been characterized during human pre-implantation development. RESULTS: We systematically analyze polyA+ messenger RNAs (mRNAs) and polyA- RNAs in individual human oocytes and pre-implantation embryos using SUPeR-seq. We de novo identify 10,032 circRNAs from 2974 hosting genes. Most of these circRNAs are developmentally stage-specific and dynamically regulated. Many of them are maternally expressed, implying their potentially important regulatory functions in oogenesis and the formation of totipotent zygotes. Comparison between human and mouse embryos reveals both high conservation and clear distinction between these two species. Human pre-implantation embryos generate more types of circRNA compared with mouse embryos and this is associated with a striking increase of the length of the circRNA flanking introns in humans. We also perform RNA de novo assembly and identify novel transcript units, many of which are potentially novel long non-coding RNAs. CONCLUSIONS: This study reports the first analysis of the whole transcriptome comprising both polyA+ mRNAs and polyA- RNAs including circRNAs during human pre-implantation development. It provides an invaluable resource for analyzing the unique function and complex regulatory mechanisms of circRNAs during this process.

Histone methyltransferase SMYD3 regulates the expression of transcriptional factors during bovine oocyte maturation and early embryonic development.

Mammalian early embryonic development is controlled by a unique program of gene expression, and involves epigenetic reprogramming of histone modifications and DNA methylation. SET and MYND domain-containing protein 3 (SMYD3) is a histone H3 lysine 4 methyltransferase that plays important roles in transcription regulation. The expression of SMYD3 has been studied in some cancer cell lines. However, its expression in oocytes and embryos has not previously been reported. Here, we detected the SMYD3 mRNA and found that it was expressed throughout bovine oocyte in vitro maturation and early embryonic development. Microinjection of SMYD3 siRNA at germinal vesicle stage decreased the transcription level of NANOG, and blocked the development of in vitro fertilization embryos at 4-8 cell stage. Conversely, Microinjection of SMYD3 siRNA at pronuclear stage did not affect early embryonic development. Our findings suggest that SMYD3 regulates the expression of NANOG, and plays an essential role in bovine early embryonic development.

Dynamic changes of histone H3 lysine 9 following trimethylation in bovine oocytes and pre-implantation embryos.

OBJECTIVES: We have examined dynamic changes of histone H3 lysine 9 following trimethylation (H3K9me3), the mRNA expression levels of SUV39H1 and SUV39H2 in bovine oocytes and the role in the development of in vitro fertilization (IVF) pre-implantation embryos. RESULTS: There were strong H3K9me3 signals in germinal vesicle (GV) oocytes but no signals in MII oocytes. H3K9me3 signals were maintained during IVF pre-implantation embryo development. SUV39H1 and SUV39H2 showed significantly higher mRNA expression levels in GV oocytes than MII oocytes (P < 0.01). SUV39H1 showed high mRNA expression level in two-cell embryos, however, SUV39H2 showed high mRNA expression level in four-cell embryos. In other development stage, SUV39H1 and SUV39H2 showed low expression levels. CONCLUSION: Bovine IVF pre-implantation embryos maintain strong H3K9me3 signals and SUV39H1 and SUV39H2 are highly expressed at the early development stage of pre-implantation embryos.

Effects of alcohol on H3K9 acetylation in mouse pre-implantation embryos.

It is well known that excessive long-term alcohol consumption is harmful, especially in pregnant women. In the present study, the Kunming white mouse was used as an animal model and indirect immunofluorescence was performed to analyze the toxic effects of alcohol on early pre-implantation embryos. H3K9 acetylation immunofluorescence could not be detected in MII oocytes. H3K9 acetylation levels in the treatment group were higher than in the control group during the morula stage, and contrary to results during the blastocyst stage. Other stages showed no obvious differences for in vivo embryos. For in vitro embryos, almost no difference was found between the two experimental groups across all stages, and both groups showed increasing H3K9 acetylation levels (except at the 2-cell stage). This study shows that H3K9 acetylation levels in early pre-implantation embryos are notably impacted by excessive alcohol ingestion by females. These data are the first step in understanding the epigenetic mechanism of alcohol toxicity in early pre-implantation mouse embryos.

Cyclosporine A improves adhesion and invasion of mouse preimplantation embryos via upregulating integrin ß3 and matrix metalloproteinase-9.

Our previous study has demonstrated cyclosporin A (CsA) promotes the migration and invasiveness of human first-trimester trophoblast cells in vitro. Here, we further investigated the effect of CsA on the early implantation in vitro of mouse embryo. Female C57 mice were superovulated and mated, and then two-cell embryos were harvested from the oviducts and sequentially cultured in vitro in G1 and G2 media with 0, 0.1, 1.0 or 10 µM of CsA. Blastocyte formation, blastocyte cell number and apoptosis, embryo hatching were assessed in 4-6 dpc. The adhesion and stretching growth of hatched embryos in laminin coated dishes were evaluated from 5 dpc to 8 dpc, and the expressions of implantation serine proteinase 1 (ISP1), integrin (itg) ß3 and matrix metalloproteinase (MMP)-9 were determined by real time PCR and immunofluorescence, respectively. We showed there was no significant difference in blastocyst formation rates, hatching rates, number of whole embryonic cells, apoptotic cells, and distribution of inner cell masses (ICMs) and trophoblasts (TB) between the CsA- and control-treated groups. Expression of ISP1 mRNA was unaffected on 5 dpc. After hatching, adhesion rate of 7 dpc significantly increased in 0.1 and 1.0 µM of CsA treatment, and embryo area of 8 dpc stretch growing on laminin were increased in 1.0 µM of CsA. The mRNA and protein expression of itgß3 and MMP-9 on 7 dpc blastocyst were up-regulated. In conclusion, CsA in low dosage up-regulates itgß3 and MMP-9 expression, and enhances embryonic adhesion and invasion, which is beneficial to the embryo implantation.