Delayed implantation (embryonic diapause) occurs when the embryo at the blastocyst stage achieves a state of suspended animation. During this period, blastocyst growth is very slow, with minimal or no cell division. Nearly 100 mammals in seven different orders undergo delayed implantation, but the underlying molecular mechanisms that direct this process remain largely unknown. In mice, ovariectomy before preimplantation ovarian estrogen secretion on day 4 of pregnancy initiates blastocyst dormancy, which normally lasts for 1-2 weeks by continued progesterone treatment, although blastocyst survival decreases with time. An estrogen injection rapidly activates blastocysts and initiates their implantation in the progesterone-primed uterus. Using this model, here we show that among Ϸ20,000 genes examined, only 229 are differentially expressed between dormant and activated blastocysts. The major functional categories of altered genes include the cell cycle, cell signaling, and energy metabolic pathways, particularly highlighting the importance of heparin-binding epidermal growth factor-like signaling in blastocyst-uterine crosstalk in implantation. The results provide evidence that the two different physiological states of the blastocyst, dormancy and activation, are molecularly distinguishable in a global perspective and underscore the importance of specific molecular pathways in these processes. This study has identified candidate genes that provide a scope for in-depth analysis of their functions and an opportunity for examining their relevance to blastocyst dormancy and activation in numerous other species for which microarray analysis is not available or possible due to very limited availability of blastocysts.S uccessful implantation results from reciprocal interactions between an implantation-competent blastocyst and a receptive uterus. Highly coordinated cellular and molecular events, directed by ovarian estrogen and progesterone (P 4 ), produce a favorable uterine environment, the receptive state, to support implantation. The blastocyst also functions as an active unit with its own molecular program of cell growth and differentiation (1, 2). It is difficult to distinguish embryonic and uterine events during normal pregnancy with respect to blastocyst activation and uterine receptivity because of the changing levels of ovarian hormones. Because estrogen is essential for on-time uterine receptivity and blastocyst activation in mice (3), ovariectomy before preimplantation estrogen secretion on the morning of day 4 results in implantation failure, initiating a state of blastocyst dormancy. This condition, referred as delayed implantation, can be maintained by continued P 4 treatment. However, implantation with blastocyst activation rapidly occurs by an estrogen injection in P 4 -primed mice (3, 4). Delayed implantation also occurs naturally (facultative) during lactation after postpartum ovulation and successful mating in mice; implantation occurs after termination of the suckling stimulus (5). Lactational delay ...

Genes encoding components of the hedgehog signaling pathway are dynamically expressed in the mouse uterus preparing for implantation. Indian hedgehog (Ihh), patched (Ptc), and Gli3 are expressed at low levels in the endometrial epithelium on day 1 of pregnancy. Transcription of Ihh increases dramatically in the luminal epithelium and glands from day 3, reaching very high levels on day 4. Over the same period, Ptc, Gli1, Gli2, and noggin are strongly upregulated in the underlying mesenchymal stroma. Transcription of Ihh in ovariectomized mice is induced by progesterone but not by estrogen. Lower induction of Ihh, Ptc, and Hoxa10 is seen in response to progesterone in the uteri of Pgr(-/-) mutant mice lacking progesterone nuclear steroid receptor. This finding suggests that the hormone may regulate Ihh through both nuclear receptor-dependent and -independent pathways. We describe a method for culturing uterine explants in the absence of epithelium. Under these conditions, recombinant N-SHH protein promotes the proliferation of mesenchyme cells and the expression of noggin. We propose that IHH made by the epithelium normally functions as a paracrine growth factor for stromal cells during the early stages of pregnancy.

Infertility and spontaneous pregnancy losses are an enduring problem to women's health. The establishment of pregnancy depends on successful implantation, where a complex series of interactions occurs between the heterogeneous cell types of the uterus and blastocyst. Although a number of genes are implicated in embryo-uterine interactions during implantation, genetic evidence suggests that only a small number of them are critical to this process. To obtain a global view and identify novel pathways of implantation, we used a dual screening strategy to analyze the expression of nearly 10,000 mouse genes by microarray analysis. Comparison of implantation and interimplantation sites by a conservative statistical approach revealed 36 up-regulated genes and 27 down-regulated genes at the implantation site. We also compared the uterine gene expression profile of progesterone-treated, delayed implanting mice to that of mice in which delayed implantation was terminated by estrogen. The results show up-regulation of 128 genes and down-regulation of 101 genes after termination of the delayed implantation. A combined analysis of these experiments showed specific up-regulation of 27 genes both at the implantation site and during uterine activation, representing a broad diversity of molecular functions. In contrast, the majority of genes that were decreased in the combined analysis were related to host immunity or the immune response, suggesting the importance of these genes in regulating the uterine environment for the implanting blastocyst. Collectively, we identified genes with recognized roles in implantation, genes with potential roles in this process, and genes whose functions have yet to be defined in this event. The identification of unique genetic markers for the onset of implantation signifies that genome-wide analysis coupled with functional assays is a promising approach to resolve the molecular pathways required for successful implantation.

Increased vascular permeability and angiogenesis at the site of blastocyst apposition in the uterus are two hallmarks of the implantation process. The present investigation shows that although the proangiogenic vascular endothelial growth factor (VEGF) and its receptor, Flk-1, are primarily important for uterine vascular permeability and angiogenesis prior to and during the attachment phase of the implantation process, VEGF in complementation with the angiopoietins and their receptor, Tie-2, directs angiogenesis during decidualization following implantation. Mice with null mutation for the gene encoding cyclooxygenase-2 (COX-2), a rate-limiting enzyme in prostaglandin (PG) biosynthesis, show implantation and decidualization failure. Using reporter and mutant mice, we show here that COX-2-derived prostaglandins (PGs) are important for uterine vascular permeability and angiogenesis during implantation and decidualization, suggesting that one cause of the failure of these latter processes in Cox-2(؊/؊) mice is the deregulated vascular events in the absence of COX-2. The attenuation of uterine angiogenesis in these mice is primarily due to defective VEGF signaling and not due to the defective angiopoietin system.

Mammalian fertility absolutely depends on synchronized development of the blastocyst to the stage when it is competent to implant, and the uterus to the stage when it is receptive to implantation. However, the molecular basis for the reciprocal interaction between the embryo and the uterus remains largely unexplored. One potentially important mechanism involves signaling between an evolutionarily conserved G protein-coupled protein cannabinoid receptor, CB1, that is expressed at high levels on the surface of the trophectoderm and anandamide (N-arachidonoylethanolamine), an endocannabinoid ligand found to be produced at higher levels by the uterus before implantation and then down-regulated at the time of implantation. Using genetic, pharmacological, and physiological approaches, we show here that anandamide within a very narrow range regulates blastocyst function and implantation by differentially modulating mitogenactivated protein kinase signaling and Ca 2؉ channel activity via CB1 receptors. Anandamide at a low concentration (7 nM) induces extracellular regulated kinase phosphorylation and nuclear translocation in trophectoderm cells without influencing Ca 2؉ channels, and renders the blastocyst competent for implantation in the receptive uterus. In contrast, anandamide at a higher concentration (28 nM) inhibits Ca 2؉ channel activity and blastocyst competency for implantation without influencing mitogen-activated protein kinase signaling. Besides uncovering a potentially important regulatory mechanism for synchronizing blastocyst and uterine competency to implantation, this observation has high clinical relevance, because elevated levels of anandamide induce spontaneous pregnancy loss in women.

Previous observations of ovulation and fertilization defects in cyclooxygenase-2 (COX-2)-deficient mice suggested that COX-2-derived ovarian prostaglandins (PGs) participate in these events. However, the specific PG and its mode of action were unknown. Subsequent studies revealed that mice deficient in EP(2), a PGE(2)-receptor subtype, have reduced litter size, apparently resulting from poor ovulation but more dramatically from impaired fertilization. Using a superovulation regimen and in vitro culture system, we demonstrate herein that the ovulatory process, not follicular growth, oocyte maturation, or fertilization, is primarily affected in adult COX-2- or EP(2)-deficient mice. Furthermore, our results show that in vitro-matured and -fertilized eggs are capable of subsequent preimplantation development. However, severely compromised ovulation in adult COX-2- or EP(2)-deficient mice is not manifested in immature (3-wk-old) COX-2- or EP(2)-deficient mice, suggesting that the process of ovulation is more dependent on PGs in adult mice. Although the processes of implantation and decidualization are defective in COX-2(-/-) mice, our present results demonstrate that these events are normal in EP(2)-deficient mice, as determined by embryo transfer and experimentally induced decidualization. Collectively, previous and present results suggest that whereas COX-2-derived PGE(2) is essential for ovulation via activation of EP(2), COX-2-derived prostacyclin is involved in implantation and decidualization via activation of peroxisome proliferator-activated receptor delta.

Increased uterine vascular permeability and angiogenesis are hallmarks of implantation and placentation. These events are profoundly influenced by vascular endothelial growth factor (VEGF). We previously showed that VEGF isoforms and VEGF receptors are expressed in the uterus, suggesting the role of VEGF in uterine vascular permeability and angiogenesis required for implantation and decidualization. We have recently shown that estrogen promotes uterine vascular permeability but inhibits angiogenesis, whereas progesterone stimulates angiogenesis with little effect on vascular permeability. However, the mechanism of differential steroid hormonal regulation of uterine angiogenesis remains unresolved. Oxygen homeostasis is essential for cell survival and is primarily mediated by hypoxia-inducible factors (HIFs). These factors are intimately associated with vascular events and induce VEGF expression by binding to the hypoxia response element in the VEGF promoter. HIF␣ isoforms function by forming heterodimers with the aryl hydrocarbon nuclear translocator (ARNT) (HIF-␤) family members. There is very limited information on the relationship among HIFs, ARNTs, and VEGF in the uterus during early pregnancy, although the role of HIFs in regulating VEGF and angiogenesis in cancers is well documented. Using molecular and physiological approaches, we here show that uterine expression of HIFs and ARNTs does not correlate with VEGF expression during the preimplantation period (days 1-4) in mice. In contrast, their expression follows the localization of uterine VEGF expression with increasing angiogenesis during the postimplantation period (days 5-8). This disparate pattern of uterine HIFs, ARNTs, and VEGF expression on days 1-4 of pregnancy suggests HIFs have multiple roles in addition to the regulation of angiogenesis during the peri-implantation period. Using pharmacological, molecular, and genetic approaches, we also observed that although progesterone primarily up-regulates uterine HIF-1␣ expression, estrogen transiently stimulates that of HIF-2␣.

The transient synthesis and accumulation of hyaluronan (HA), an extracellular matrix component of cumulus cells, brings about expansion of cumulus-oocyte complexes (COCs) in preovulatory mammalian follicles. In this study, we investigated the mRNA expressions of hyaluronan synthase 2 (has2), hyaluronan synthase 3 (has3), and CD44, as well as the responsiveness to eCG and porcine follicular fluid (pFF) of these genes, in porcine COCs, oocytectomized complexes (OXCs), and oocytes during in vitro maturation. Immunolocalization of CD44 was also analyzed in COCs. After 12 h of culture, the area of cumulus expansion in medium 199 supplemented with both 10 IU/ml eCG and 10% (v/v) pFF was significantly greater than that in the medium supplemented with eCG or pFF. Oocytectomy reduced the expansion area in the group supplemented with eCG. In reverse transcription-polymerase chain reaction analysis, all transcripts were identified in COCs, but has3 transcript was not found in OXCs. Only has3 mRNA was detectable in oocytes, indicating that cumulus cells express has2 and CD44 mRNAs, and oocytes express has3 mRNA. The expression levels of has2 and CD44 mRNAs in COCs and OXCs increased in the presence of eCG and pFF after 24 h of culture, suggesting that these genes have a positive dependency on eCG and pFF. In contrast, the high level of has3 mRNA was detected in COCs cultured in the medium alone. Oocytectomy slightly reduced the expression level of has2 mRNA. On immunostaining for CD44, CD44 was expressed apparently in COCs cultured with eCG and pFF for 24 h. The positive staining was distributed on cytoplasm along the perimembrane of cumulus cells and at the junctions between cumulus cells and oocytes. CD44 was also localized on cytoplasm of some oocytes. These results indicate that 1) porcine oocytes promote eCG-dependent cumulus expansion and the expression of has2 mRNA in cumulus cells, but these are not essential for expansion of cumulus cells and the expression of has2 mRNA; 2) HAS2 is involved in HA synthesis during cumulus expansion, and eCG and pFF up-regulate its expression; 3) the expression profile of the has3 mRNA that is transcribed in oocytes is different from those of has2 and CD44 mRNA; and 4) CD44 may participate in the interaction between cumulus cells and oocytes.